Fixed dosing of her antibodies

ABSTRACT

The present invention concerns fixed dosing of HER antibodies, such as Pertuzumab.

This is a continuation application which claims priority under 35 USC§120 to continuation application Ser. No. 13/744,591, filed Jan. 18,2013, which claims priority to divisional application Ser. No.12/248,223, filed Oct. 9, 2008 (now U.S. Pat. No. 8,404,234), whichclaims priority to non-provisional application Ser. No. 11/154,091,filed Jun. 15, 2005 (now U.S. Pat. No. 7,449,184), which claims priorityunder 35 USC §119 to provisional application 60/645,697, filed Jan. 21,2005, the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention concerns fixed dosing of HER antibodies, such aspertuzumab.

BACKGROUND OF THE INVENTION HER Receptors and Antibodies Thereagainst

The HER family of receptor tyrosine kinases are important mediators ofcell growth, differentiation and survival. The receptor family includesfour distinct members including epidermal growth factor receptor (EGFR,ErbB1, or HER1), HER2 (ErbB2 or p185^(neu)), HER3 (ErbB3) and HER4(ErbB4 or tyro2).

EGFR, encoded by the erbB1 gene, has been causally implicated in humanmalignancy. In particular, increased expression of EGFR has beenobserved in breast, bladder, lung, head, neck and stomach cancer as wellas glioblastomas. Increased EGFR receptor expression is often associatedwith increased production of the EGFR ligand, transforming growth factoralpha (TGF-α), by the same tumor cells resulting in receptor activationby an autocrine stimulatory pathway. Baselga and Mendelsohn, Pharmac.Ther. 64:127-154 (1994). Monoclonal antibodies directed against the EGFRor its ligands, TGF-α and EGF, have been evaluated as therapeutic agentsin the treatment of such malignancies. See, e.g., Baselga andMendelsohn, supra; Masui et al. Cancer Research 44:1002-1007 (1984); andWu et al. J. Clin. Invest. 95:1897-1905 (1995).

The second member of the HER family, p185^(neu), was originallyidentified as the product of the transforming gene from neuroblastomasof chemically treated rats. The activated form of the neu proto-oncogeneresults from a point mutation (valine to glutamic acid) in thetransmembrane region of the encoded protein. Amplification of the humanhomolog of neu is observed in breast and ovarian cancers and correlateswith a poor prognosis (Slamon et al., Science, 235:177-182 (1987);Slamon et al., Science, 244:707-712 (1989); and U.S. Pat. No.4,968,603). To date, no point mutation analogous to that in the neuproto-oncogene has been reported for human tumors. Overexpression ofHER2 (frequently but not uniformly due to gene amplification) has alsobeen observed in other carcinomas including carcinomas of the stomach,endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas andbladder. See, among others, King et al., Science, 229:974 (1985); Yokotaet al., Lancet: 1:765-767 (1986); Fukushige et al., Mol Cell Biol.,6:955-958 (1986); Guerin et al., Oncogene Res., 3:21-31 (1988); Cohen etal., Oncogene, 4:81-88 (1989); Yonemura et al., Cancer Res., 51:1034(1991); Borst et al., Gynecol. Oncol., 38:364 (1990); Weiner et al.,Cancer Res., 50:421-425 (1990); Kern et al., Cancer Res., 50:5184(1990); Park et al., Cancer Res., 49:6605 (1989); Zhau et al., Mol.Carcinog., 3:254-257 (1990); Aasland et al. Br. J. Cancer 57:358-363(1988); Williams et al., Pathobiology 59:46-52 (1991); and McCann etal., Cancer, 65:88-92 (1990). HER2 may be overexpressed in prostatecancer (Gu et al., Cancer Lett. 99:185-9 (1996); Ross et al., Hum.Pathol. 28:827-33 (1997); Ross et al., Cancer 79:2162-70 (1997); andSadasivan et al., J. Urol. 150:126-31 (1993)).

Antibodies directed against the rat p185^(neu) and human HER2 proteinproducts have been described.

Drebin and colleagues have raised antibodies against the rat neu geneproduct, p185^(neu) See, for example, Drebin et al., Cell 41:695-706(1985); Myers et al., Meth. Enzym. 198:277-290 (1991); and WO94/22478.Drebin et al. Oncogene 2:273-277 (1988) report that mixtures ofantibodies reactive with two distinct regions of p185^(neu) result insynergistic anti-tumor effects on neu-transformed NIH-3T3 cellsimplanted into nude mice. See also U.S. Pat. No. 5,824,311 issued Oct.20, 1998.

Hudziak et al., Mol. Cell. Biol. 9(3):1165-1172 (1989) describe thegeneration of a panel of HER2 antibodies which were characterized usingthe human breast tumor cell line SK-BR-3. Relative cell proliferation ofthe SK-BR-3 cells following exposure to the antibodies was determined bycrystal violet staining of the monolayers after 72 hours. Using thisassay, maximum inhibition was obtained with the antibody called 4D5which inhibited cellular proliferation by 56%. Other antibodies in thepanel reduced cellular proliferation to a lesser extent in this assay.The antibody 4D5 was further found to sensitize HER2-overexpressingbreast tumor cell lines to the cytotoxic effects of TNF-α. See also U.S.Pat. No. 5,677,171 issued Oct. 14, 1997. The HER2 antibodies discussedin Hudziak et al. are further characterized in Fendly et al. CancerResearch 50:1550-1558 (1990); Kotts et al. In Vitro 26(3):59A (1990);Sarup et al. Growth Regulation 1:72-82 (1991); Shepard et al. J. Clin.Immunol. 11(3):117-127 (1991); Kumar et al. Mol. Cell. Biol.11(2):979-986 (1991); Lewis et al. Cancer Immunol. Immunother.37:255-263 (1993); Pietras et al. Oncogene 9:1829-1838 (1994); Vitettaet al. Cancer Research 54:5301-5309 (1994); Sliwkowski et al. J. Biol.Chem. 269(20):14661-14665 (1994); Scott et al. J. Biol. Chem.266:14300-5 (1991); D′souza et al. Proc. Natl. Acad. Sci. 91:7202-7206(1994); Lewis et al. Cancer Research 56:1457-1465 (1996); and Schaeferet al. Oncogene 15:1385-1394 (1997).

A recombinant humanized version of the murine HER2 antibody 4D5(huMAb4D5-8, rhuMAb HER2, trastuzumab or HERCEPTIN®; U.S. Pat. No.5,821,337) is clinically active in patients with HER2-overexpressingmetastatic breast cancers that have received extensive prior anti-cancertherapy (Baselga et al., J. Clin. Oncol. 14:737-744 (1996)). Trastuzumabreceived marketing approval from the Food and Drug Administration Sep.25, 1998 for the treatment of patients with metastatic breast cancerwhose tumors overexpress the HER2 protein.

Other HER2 antibodies with various properties have been described inTagliabue et al. Int. J. Cancer 47:933-937 (1991); McKenzie et al.Oncogene 4:543-548 (1989); Maier et al. Cancer Res. 51:5361-5369 (1991);Bacus et al. Molecular Carcinogenesis 3:350-362 (1990); Stancovski etal. PNAS (USA) 88:8691-8695 (1991); Bacus et al. Cancer Research52:2580-2589 (1992); Xu et al. Int. J. Cancer 53:401-408 (1993);WO94/00136; Kasprzyk et al. Cancer Research 52:2771-2776 (1992); Hancocket al. Cancer Res. 51:4575-4580 (1991); Shawver et al. Cancer Res.54:1367-1373 (1994); Arteaga et al. Cancer Res. 54:3758-3765 (1994);Harwerth et al. J. Biol. Chem. 267:15160-15167 (1992); U.S. Pat. No.5,783,186; and Klapper et al. Oncogene 14:2099-2109 (1997).

Homology screening has resulted in the identification of two other HERreceptor family members; HER3 (U.S. Pat. Nos. 5,183,884 and 5,480,968 aswell as Kraus et al., PNAS (USA) 86:9193-9197 (1989)) and HER4 (EP PatAppln No 599,274; Plowman et al., Proc. Natl. Acad. Sci. USA,90:1746-1750 (1993); and Plowman et al., Nature, 366:473-475 (1993)).Both of these receptors display increased expression on at least somebreast cancer cell lines.

The HER receptors are generally found in various combinations in cellsand heterodimerization is thought to increase the diversity of cellularresponses to a variety of HER ligands (Earp et al. Breast CancerResearch and Treatment 35: 115-132 (1995)). EGFR is bound by sixdifferent ligands; epidermal growth factor (EGF), transforming growthfactor alpha (TGF-α), amphiregulin, heparin binding epidermal growthfactor (HB-EGF), betacellulin and epiregulin (Groenen et al. GrowthFactors 11:235-257 (1994)). A family of heregulin proteins resultingfrom alternative splicing of a single gene are ligands for HER3 andHER4. The heregulin family includes alpha, beta and gamma heregulins(Holmes et al., Science, 256:1205-1210 (1992); U.S. Pat. No. 5,641,869;and Schaefer et al. Oncogene 15:1385-1394 (1997)); neu differentiationfactors (NDFs), glial growth factors (GGFs); acetylcholine receptorinducing activity (ARIA); and sensory and motor neuron derived factor(SMDF). For a review, see Groenen et al. Growth Factors 11:235-257(1994); Lemke, G. Molec. & Cell. Neurosci. 7:247-262 (1996) and Lee etal. Pharm. Rev. 47:51-85 (1995). Recently three additional HER ligandswere identified; neuregulin-2 (NRG-2) which is reported to bind eitherHER3 or HER4 (Chang et al. Nature 387 509-512 (1997); and Carraway et alNature 387:512-516 (1997)); neuregulin-3 which binds HER4 (Zhang et al.PNAS (USA) 94(18):9562-7 (1997)); and neuregulin-4 which binds HER4(Harari et al. Oncogene 18:2681-89 (1999)) HB-EGF, betacellulin andepiregulin also bind to HER4.

While EGF and TGFα do not bind HER2, EGF stimulates EGFR and HER2 toform a heterodimer, which activates EGFR and results intransphosphorylation of HER2 in the heterodimer. Dimerization and/ortransphosphorylation appears to activate the HER2 tyrosine kinase. SeeEarp et al., supra. Likewise, when HER3 is co-expressed with HER2, anactive signaling complex is formed and antibodies directed against HER2are capable of disrupting this complex (Sliwkowski et al., J. Biol.Chem., 269(20):14661-14665 (1994)). Additionally, the affinity of HER3for heregulin (HRG) is increased to a higher affinity state whenco-expressed with HER2. See also, Levi et al., Journal of Neuroscience15: 1329-1340 (1995); Morrissey et al., Proc. Natl. Acad. Sci. USA 92:1431-1435 (1995); and Lewis et al., Cancer Res., 56:1457-1465 (1996)with respect to the HER2-HER3 protein complex. HER4, like HER3, forms anactive signaling complex with HER2 (Carraway and Cantley, Cell 78:5-8(1994)).

Patent publications related to HER antibodies include: U.S. Pat. No.5,677,171, U.S. Pat. No. 5,720,937, U.S. Pat. No. 5,720,954, U.S. Pat.No. 5,725,856, U.S. Pat. No. 5,770,195, U.S. Pat. No. 5,772,997, U.S.Pat. No. 6,165,464, U.S. Pat. No. 6,387,371, U.S. Pat. No. 6,399,063,US2002/0192211A1, U.S. Pat. No. 6,015,567, U.S. Pat. No. 6,333,169, U.S.Pat. No. 4,968,603, U.S. Pat. No. 5,821,337, U.S. Pat. No. 6,054,297,U.S. Pat. No. 6,407,213, U.S. Pat. No. 6,719,971, U.S. Pat. No.6,800,738, US2004/0236078A1, U.S. Pat. No. 5,648,237, U.S. Pat. No.6,267,958, U.S. Pat. No. 6,685,940, U.S. Pat. No. 6,821,515, WO98/17797,U.S. Pat. No. 6,127,526, U.S. Pat. No. 6,333,398, U.S. Pat. No.6,797,814, U.S. Pat. No. 6,339,142, U.S. Pat. No. 6,417,335, U.S. Pat.No. 6,489,447, WO99/31140, US2003/0147884A1, US2003/0170234A1,US2005/0002928A1, U.S. Pat. No. 6,573,043, US2003/0152987A1, WO99/48527,US2002/0141993A1, WO01/00245, US2003/0086924, US2004/0013667A1,WO00/69460, WO01/00238, WO01/15730, U.S. Pat. No. 6,627,196B1, U.S. Pat.No. 6,632,979B1, WO01/00244, US2002/0090662A1, WO01/89566,US2002/0064785, US2003/0134344, WO 04/24866, US2004/0082047,US2003/0175845A1, WO03/087131, US2003/0228663, WO2004/008099A2,US2004/0106161, WO2004/048525, US2004/0258685A1, U.S. Pat. No.5,985,553, U.S. Pat. No. 5,747,261, U.S. Pat. No. 4,935,341, U.S. Pat.No. 5,401,638, U.S. Pat. No. 5,604,107, WO 87/07646, WO 89/10412, WO91/05264, EP 412,116 B1, EP 494,135 B1, U.S. Pat. No. 5,824,311, EP444,181 B1, EP 1,006,194 A2, US 2002/0155527A1, WO 91/02062, U.S. Pat.No. 5,571,894, U.S. Pat. No. 5,939,531, EP 502,812 B1, WO 93/03741, EP554,441 B1, EP 656,367 A1, U.S. Pat. No. 5,288,477, U.S. Pat. No.5,514,554, U.S. Pat. No. 5,587,458, WO 93/12220, WO 93/16185, U.S. Pat.No. 5,877,305, WO 93/21319, WO 93/21232, U.S. Pat. No. 5,856,089, WO94/22478, U.S. Pat. No. 5,910,486, U.S. Pat. No. 6,028,059, WO 96/07321,U.S. Pat. No. 5,804,396, U.S. Pat. No. 5,846,749, EP 711,565, WO96/16673, U.S. Pat. No. 5,783,404, U.S. Pat. No. 5,977,322, U.S. Pat.No. 6,512,097, WO 97/00271, U.S. Pat. No. 6,270,765, U.S. Pat. No.6,395,272, U.S. Pat. No. 5,837,243, WO 96/40789, U.S. Pat. No.5,783,186, U.S. Pat. No. 6,458,356, WO 97/20858, WO 97/38731, U.S. Pat.No. 6,214,388, U.S. Pat. No. 5,925,519, WO 98/02463, U.S. Pat. No.5,922,845, WO 98/18489, WO 98/33914, U.S. Pat. No. 5,994,071, WO98/45479, U.S. Pat. No. 6,358,682 B1, US 2003/0059790, WO 99/55367, WO01/20033, US 2002/0076695 A1, WO 00/78347, WO 01/09187, WO 01/21192, WO01/32155, WO 01/53354, WO 01/56604, WO 01/76630, WO02/05791, WO02/11677, U.S. Pat. No. 6,582,919, US2002/0192652A1, US 2003/0211530A1,WO 02/44413, US 2002/0142328, U.S. Pat. No. 6,602,670 B2, WO 02/45653,WO 02/055106, US 2003/0152572, US 2003/0165840, WO 02/087619, WO03/006509, WO03/012072, WO 03/028638, US 2003/0068318, WO 03/041736, EP1,357,132, US 2003/0202973, US 2004/0138160, U.S. Pat. No. 5,705,157,U.S. Pat. No. 6,123,939, EP 616,812 B1, US 2003/0103973, US2003/0108545, U.S. Pat. No. 6,403,630 B1, WO 00/61145, WO 00/61185, U.S.Pat. No. 6,333,348 B1, WO 01/05425, WO 01/64246, US 2003/0022918, US2002/0051785 A1, U.S. Pat. No. 6,767,541, WO 01/76586, US 2003/0144252,WO 01/87336, US 2002/0031515 A1, WO 01/87334, WO 02/05791, WO 02/09754,US 2003/0157097, US 2002/0076408, WO 02/055106, WO 02/070008, WO02/089842 and WO 03/86467.

Diagnostics

Patients treated with the HER2 antibody trastuzumab are selected fortherapy based on HER2 overexpression/amplification. See, for example,WO99/31140 (Paton et al.), US2003/0170234A1 (Hellmann, S.), andUS2003/0147884 (Paton et al.); as well as WO01/89566, US2002/0064785,and US2003/0134344 (Mass et al.). See, also, US2003/0152987, Cohen etal., concerning immunohistochemistry (1HC) and fluorescence in situhybridization (FISH) for detecting HER2 overexpression andamplification.

WO2004/053497 (Bacus et al.) refers to determining or predictingresponse to HERCEPTIN® therapy. US2004/013297A1 (Bacus et al.) concernsdetermining or predicting response to ABX0303 EGFR antibody therapy.WO2004/000094 (Bacus et al.) is directed to determining response toGW572016, a small molecule, EGFR-HER2 tyrosine kinase inhibitor.WO2004/063709, Amler et al., refers to biomarkers and methods fordetermining sensitivity to EGFR inhibitor, erlotinib HCl.US2004/0209290, Cobleigh et al., concerns gene expression markers forbreast cancer prognosis.

Patients treated with pertuzumab can be selected for therapy based onHER activation or dimerization. Patent publications concerningpertuzumab and selection of patients for therapy therewith include:WO01/00245 (Adams et al.); US2003/0086924 (Sliwkowski, M.);US2004/0013667A1 (Sliwkowski, M.); as well as WO2004/008099A2, andUS2004/0106161 (Bossenmaier et al.).

Cronin et al., Am. J. Path. 164(1): 35-42 (2004) describes measurementof gene expression in archival paraffin-embedded tissues. Ma et al.Cancer Cell 5:607-616 (2004) describes gene profiling by geneoliogonucleotide microarray using isolated RNA from tumor-tissuesections taken from archived primary biopsies.

Dosing of Anticancer Drugs and HER Antibodies

Papers discussing dosing of anticancer drugs include: Egorin, M. J ClinOncol 2003; 21:182-3 (2003); Baker et al. J Natl Cancer Inst 94:1883-8(2002); Felici et al. Eur J Cancer 38:1677-84 (2002); Loos et al. Clin.Cancer Res. 6:2685-9 (2000); de Jongh et al. J. Clin Oncol. 19:3733-9(2001); Mathijssen et al. J. Clin Oncol. 20:81-7 (2002); and de Jong etal. Clin Cancer Res 10:4068-71 (2004).

Typically, commercially available humanized IgG monoclonal antibodies(i.e. trastuzumab, and bevacizumab, Genentech Inc., South San Franciscoand gemtuzumab ozogomicin, Wyeth Pharmaceuticals, Philadelphia) andcytotoxic small molecule drugs in oncology have been administered on aweight-based (mg/kg) or body surface area-based (BSA) dosing method.

Cetuximab (ERBITUX®) is an antibody that binds EGF receptor and isapproved for therapy of colorectal cancer. In colorectal cancer,cetuximab 400 mg/m2 is given as a loading dose by intravenous infusionover 2 hours. This is followed by once weekly maintenance doses of 250mg/m2 given over 1 hour. See cetuximab prescribing information.

Trastuzumab (HERCEPTIN®) is administered to patients with metastaticbreast cancer as a 4 mg/kg loading dose, followed by weekly 2 mg/kgdoses. See trastuzumab prescribing information.

See, also, WO99/31140; US2003/0147884A1; US2003/0170234A1;US2005/0002928A1; WO00/69460; WO01/15730 and U.S. Pat. No. 6,627,196B1concerning trastuzumab dosing.

Pertuzumab (also known as recombinant human monoclonal antibody 2C4;OMNITARG™, Genentech, Inc, South San Francisco) represents the first ina new class of agents known as HER dimerization inhibitors (HDI) andfunctions to inhibit the ability of HER2 to form active heterodimerswith other HER receptors (such as EGFR/HER1, HER3 and HER4) and isactive irrespective of HER2 expression levels. See, for example, Harariand Yarden Oncogene 19:6102-14 (2000); Yarden and Sliwkowski. Nat. RevMol Cell Biol 2:127-37 (2001); Sliwkowski Nat Struct Biol 10:158-9(2003); Cho et al. Nature 421:756-60 (2003); and Malik et al. Pro AmAssoc Cancer Res 44:176-7 (2003).

Pertuzumab blockade of the formation of HER2-HER3 heterodimers in tumorcells has been demonstrated to inhibit critical cell signaling, whichresults in reduced tumor proliferation and survival (Agus et al. CancerCell 2:127-37 (2002)).

Pertuzumab has undergone testing as a single agent in the clinic with aphase Ia trial in patients with advanced cancers and phase II trials inpatients with ovarian cancer and breast cancer as well as lung andprostate cancer. In a Phase I study, patients with incurable, locallyadvanced, recurrent or metastatic solid tumors that had progressedduring or after standard therapy were treated with pertuzumab givenintravenously every 3 weeks. Pertuzumab was generally well tolerated.Tumor regression was achieved in 3 of 20 patients evaluable forresponse. Two patients had confirmed partial responses. Stable diseaselasting for more than 2.5 months was observed in 6 of 21 patients (Aguset al. Pro Am Assoc Cancer Res 22:192 (2003)). At doses of 2.0-15 mg/kg,the pharmacokinetic of pertuzumab was linear, and mean clearance rangedfrom 2.69 to 3.74 mL/day/kg and the mean terminal elimination half-liferanged from 15.3 to 27.6 days. Antibodies to pertuzumab were notdetected (Allison et al. Pro Am Soc Clin Oncol 22:197 (2003)).Pertuzumab was dosed on a weight-basis (mg/kg) in the Phase I trial.Phase II trials have been initiated using a fixed-dose.

SUMMARY OF THE INVENTION

The present invention provides the first critical assessment of theimpact and utility of fixed dosing of a humanized IgG1 monoclonalantibody on pharmacokinetics and target drug concentrations. The primaryobjectives of this analysis of the HER antibody pertuzumab were to: 1)evaluate the population pharmacokinetic and predictive covariates forpertuzumab in cancer patients, and 2) examine the variability ofsteady-state trough concentrations and exposures after fixed, or bodyweight-, and body surface area (BSA)-based dosing.

Accordingly, in a first aspect, the invention provides a method fortreating cancer comprising administering one or more fixed dose(s) of aHER antibody to a human patient in an amount effective to treat thecancer.

In another aspect, the invention provides a method of treating cancer ina human patient comprising administering at least one fixed dose ofpertuzumab to the patient, wherein the fixed dose is selected from thegroup consisting of approximately 420 mg, approximately 525 mg,approximately 840 mg, and approximately 1050 mg of pertuzumab.

The invention also concerns an article of manufacture comprising a vialcontaining a fixed dose of a HER antibody, wherein the fixed dose isselected from the group consisting of approximately 420 mg,approximately 525 mg, approximately 840 mg, and approximately 1050 mg ofthe HER antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic of the HER2 protein structure, and aminoacid sequences for Domains I-IV (SEQ ID Nos.19-22, respectively) of theextracellular domain thereof.

FIGS. 2A and 2B depict alignments of the amino acid sequences of thevariable light (V_(L)) (FIG. 2A) and variable heavy (V_(H)) (FIG. 2B)domains of murine monoclonal antibody 2C4 (SEQ ID Nos. 1 and 2,respectively); V_(L) and V_(H) domains of humanized 2C4 version 574 (SEQID Nos. 3 and 4, respectively), and human V_(L) and V_(H) consensusframeworks (hum κ1, light kappa subgroup I; humIII, heavy subgroup III)(SEQ ID Nos. 5 and 6, respectively). Asterisks identify differencesbetween humanized 2C4 version 574 and murine monoclonal antibody 2C4 orbetween humanized 2C4 version 574 and the human framework.Complementarity Determining Regions (CDRs) are in brackets.

FIGS. 3A and 3B show the amino acid sequences of pertuzumab light chainand heavy chain (SEQ ID Nos. 13 and 14, respectively). CDRs are shown inbold. Calculated molecular mass of the light chain and heavy chain are23,526.22 Da and 49,216.56 Da (cysteines in reduced form). Thecarbohydrate moiety is attached to Asn 299 of the heavy chain.

FIG. 4 depicts, schematically, binding of 2C4 at the heterodimericbinding site of HER2, thereby preventing heterodimerization withactivated EGFR or HER3.

FIG. 5 depicts coupling of HER2/HER3 to the MAPK and Aid pathways.

FIG. 6 compares various activities of trastuzumab and pertuzumab.

FIGS. 7A and 7B show the amino acid sequences of trastuzumab light chain(FIG. 7A; SEQ ID No. 15) and heavy chain (FIG. 7B; SEQ ID No. 16),respectively.

FIGS. 8A and 8B depict a variant pertuzumab light chain sequence (FIG.8A; SEQ ID No. 17) and a variant pertuzumab heavy chain sequence (FIG.8B; SEQ ID No. 18), respectively.

FIGS. 9A and 9B are representative profiles of a single subject's PKdata fitted by a one- (FIG. 9A) or two- (FIG. 9B) compartmental model.Open circles indicate observed concentration. Solid and dotted linesindicate population predicted and individual predicted concentration,respectively.

FIGS. 10A and 10B represent model diagnostic plots. FIG. 10A depictsobserved versus predicted pertuzumab concentrations. The solid line isthe line of unity. FIG. 10B depicts weighted residuals versus predictedpertuzumab concentrations. The dashed line is a LOESS smooth of data.

FIGS. 11A and 11B depict random effect (η) for clearance (CL) by weight(WT) and volume at the central compartment (Vc) by body surface area(BSA) for the base model (FIG. 11A) and final model (FIG. 11B).

FIGS. 12A-F show model evaluation of pertuzumab final populationpharmacokinetic model using a posterior model check. Posteriorpredictive distribution and observed values for the test statistics:FIG. 12A-2.5^(th); FIG. 12B-5^(th); FIG. 12C-50^(th), FIG. 12D-90^(th),FIG. 12E-95^(th), FIG. 12F-97.5^(th). The vertical line on eachhistogram represents the observed value of the test statistic.

FIG. 13 illustrates the predicted pertuzumab steady state troughconcentration (Day 84) after a fixed, weight (WT) or BSA-based dose for1000 simulated subjects bootstrapped from original pharmacokinetic (PK)dataset according to the final model.

FIGS. 14A and 14B show the predicted pertuzumab steady state troughconcentration (day 84) after a fixed, weight (WT) or BSA-based dose forpatient populations with ≦10^(th) (50.4 kg) (FIG. 14A) or ≧90^(th) (88.5kg) (FIG. 14B) WT values.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

A “fixed” or “flat” dose of a therapeutic agent herein refers to a dosethat is administered to a human patient without regard for the weight(WT) or body surface area (BSA) of the patient. The fixed or flat doseis therefore not provided as a mg/kg dose or a mg/m² dose, but rather asan absolute amount of the therapeutic agent.

A “loading” dose herein generally comprises an initial dose of atherapeutic agent administered to a patient, and is followed by one ormore maintenance dose(s) thereof. Generally, a single loading dose isadministered, but multiple loading doses are contemplated herein.Usually, the amount of loading dose(s) administered exceeds the amountof the maintenance dose(s) administered and/or the loading dose(s) areadministered more frequently than the maintenance dose(s), so as toachieve the desired steady-state concentration of the therapeutic agentearlier than can be achieved with the maintenance dose(s).

A “maintenance” dose herein refers to one or more doses of a therapeuticagent administered to the patient over a treatment period. Usually, themaintenance doses are administered at spaced treatment intervals, suchas approximately every week, approximately every 2 weeks, approximatelyevery 3 weeks, or approximately every 4 weeks.

A “HER receptor” is a receptor protein tyrosine kinase which belongs tothe HER receptor family and includes EGFR, HER2, HER3 and HER4receptors. The HER receptor will generally comprise an extracellulardomain, which may bind an HER ligand and/or dimerize with another HERreceptor molecule; a lipophilic transmembrane domain; a conservedintracellular tyrosine kinase domain; and a carboxyl-terminal signalingdomain harboring several tyrosine residues which can be phosphorylated.The HER receptor may be a “native sequence” HER receptor or an “aminoacid sequence variant” thereof. Preferably the HER receptor is nativesequence human HER receptor.

The terms “ErbB1,” “HER1”, “epidermal growth factor receptor” and “EGFR”are used interchangeably herein and refer to EGFR as disclosed, forexample, in Carpenter et al. Ann. Rev. Biochem. 56:881-914 (1987),including naturally occurring mutant forms thereof (e.g. a deletionmutant EGFR as in Humphrey et al. PNAS (USA) 87:4207-4211 (1990)). erbB1refers to the gene encoding the EGFR protein product.

The expressions “ErbB2” and “HER2” are used interchangeably herein andrefer to human HER2 protein described, for example, in Semba et al.,PNAS (USA) 82:6497-6501 (1985) and Yamamoto et al. Nature 319:230-234(1986) (Genebank accession number X03363). The term “erbB2” refers tothe gene encoding human ErbB2 and “neu” refers to the gene encoding ratp185^(neu). Preferred HER2 is native sequence human HER2.

The extracellular domain of HER2 comprises four domains: “Domain I”(amino acid residues from about 1-195; SEQ ID NO:19), “Domain II” (aminoacid residues from about 196-319; SEQ ID NO:20), “Domain III” (aminoacid residues from about 320-488: SEQ ID NO:21), and “Domain IV” (aminoacid residues from about 489-630; SEQ ID NO:22) (residue numberingwithout signal peptide). See Garrett et al. Mol. Cell. 11: 495-505(2003), Cho et al. Nature 421: 756-760 (2003), Franklin et al. CancerCell 5:317-328 (2004), and Plowman et al. Proc. Natl. Acad. Sci.90:1746-1750 (1993), as well as FIG. 1 herein.

“ErbB3” and “HER3” refer to the receptor polypeptide as disclosed, forexample, in U.S. Pat. Nos. 5,183,884 and 5,480,968 as well as Kraus etal. PNAS (USA) 86:9193-9197 (1989).

The terms “ErbB4” and “HER4” herein refer to the receptor polypeptide asdisclosed, for example, in EP Pat Appln No 599,274; Plowman et al.,Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993); and Plowman et al.,Nature, 366:473-475 (1993), including isoforms thereof, e.g., asdisclosed in WO99/19488, published Apr. 22, 1999.

By “HER ligand” is meant a polypeptide which binds to and/or activates aHER receptor. The HER ligand of particular interest herein is a nativesequence human HER ligand such as epidermal growth factor (EGF) (Savageet al., J. Biol. Chem. 247:7612-7621 (1972)); transforming growth factoralpha (TGF-α) (Marquardt et al., Science 223:1079-1082 (1984));amphiregulin also known as schwanoma or keratinocyte autocrine growthfactor (Shoyab et al. Science 243:1074-1076 (1989); Kimura et al. Nature348:257-260 (1990); and Cook et al. Mol. Cell. Biol. 11:2547-2557(1991)); betacellulin (Shing et al., Science 259:1604-1607 (1993); andSasada et al. Biochem. Biophys. Res. Commun. 190:1173 (1993));heparin-binding epidermal growth factor (HB-EGF) (Higashiyama et al.,Science 251:936-939 (1991)); epiregulin (Toyoda et al., J. Biol. Chem.270:7495-7500 (1995); and Komurasaki et al. Oncogene 15:2841-2848(1997)); a heregulin (see below); neuregulin-2 (NRG-2) (Carraway et al.,Nature 387:512-516 (1997)); neuregulin-3 (NRG-3) (Zhang et al., Proc.Natl. Acad. Sci. 94:9562-9567 (1997)); neuregulin-4 (NRG-4) (Harari etal. Oncogene 18:2681-89 (1999)); and cripto (CR-1) (Kannan et al. J.Biol. Chem. 272(6):3330-3335 (1997)). HER ligands which bind EGFRinclude EGF, TGF-α, amphiregulin, betacellulin, HB-EGF and epiregulin.HER ligands which bind HER3 include heregulins. HER ligands capable ofbinding HER4 include betacellulin, epiregulin, HB-EGF, NRG-2, NRG-3,NRG-4, and heregulins.

“Heregulin” (HRG) when used herein refers to a polypeptide encoded bythe heregulin gene product as disclosed in U.S. Pat. No. 5,641,869, orMarchionni et al., Nature, 362:312-318 (1993). Examples of heregulinsinclude heregulin-α, heregulin-β, heregulin-β and heregulin-β (Holmes etal., Science, 256:1205-1210 (1992); and U.S. Pat. No. 5,641,869); neudifferentiation factor (NDF) (Peles et al. Cell 69: 205-216 (1992));acetylcholine receptor-inducing activity (ARIA) (Falls et al. Cell72:801-815 (1993)); glial growth factors (GGFs) (Marchionni et al.,Nature, 362:312-318 (1993)); sensory and motor neuron derived factor(SMDF) (Ho et al. J. Biol. Chem. 270:14523-14532 (1995)); γ-heregulin(Schaefer et al. Oncogene 15:1385-1394 (1997)).

A “HER dimer” herein is a noncovalently associated dimer comprising atleast two HER receptors. Such complexes may form when a cell expressingtwo or more HER receptors is exposed to an HER ligand and can beisolated by immunoprecipitation and analyzed by SDS-PAGE as described inSliwkowski et al., J. Biol. Chem., 269(20):14661-14665 (1994), forexample. Examples of such HER dimers include EGFR-HER2, HER2-HER3 andHER3-HER4 heterodimers. Moreover, the HER dimer may comprise two or moreHER2 receptors combined with a different HER receptor, such as HER3,HER4 or EGFR. Other proteins, such as a cytokine receptor subunit (e.g.gp130) may be associated with the dimer.

A “HER inhibitor” is an agent which interferes with HER activation orfunction. Examples of HER inhibitors include HER antibodies (e.g. EGFR,HER2, HER3, or HER4 antibodies); EGFR-targeted drugs; small molecule HERantagonists; HER tyrosine kinase inhibitors; HER2 and EGFR dual tyrosinekinase inhibitors such as lapatinib/GW572016; antisense molecules (see,for example, WO2004/87207); and/or agents that bind to, or interferewith function of, downstream signaling molecules, such as MAPK or Aid(see FIG. 5). Preferably, the HER inhibitor is an antibody or smallmolecule which binds to a HER receptor.

A “HER antibody” is an antibody that binds to a HER receptor.Optionally, the HER antibody further interferes with HER activation orfunction. Preferably, the HER antibody binds to the HER2 receptor. AHER2 antibody of particular interest herein is pertuzumab. Anotherexample of a HER2 antibody is trastuzumab. Examples of EGFR antibodiesinclude cetuximab and ABX0303.

“HER activation” refers to activation, or phosphorylation, of any one ormore HER receptors. Generally, HER activation results in signaltransduction (e.g. that caused by an intracellular kinase domain of aHER receptor phosphorylating tyrosine residues in the HER receptor or asubstrate polypeptide). HER activation may be mediated by HER ligandbinding to a HER dimer comprising the HER receptor of interest. HERligand binding to a HER dimer may activate a kinase domain of one ormore of the HER receptors in the dimer and thereby results inphosphorylation of tyrosine residues in one or more of the HER receptorsand/or phosphorylation of tyrosine residues in additional substratepolypeptides(s), such as Aid or MAPK intracellular kinases. See, FIG. 5,for example.

“Phosphorylation” refers to the addition of one or more phosphategroup(s) to a protein, such as a HER receptor, or substrate thereof.

An antibody which “inhibits HER dimerization” is an antibody whichinhibits, or interferes with, formation of a HER dimer. Preferably, suchan antibody binds to HER2 at the heterodimeric binding site thereof. Themost preferred dimerization inhibiting antibody herein is pertuzumab orMAb 2C4. Binding of 2C4 to the heterodimeric binding site of HER2 isillustrated in FIG. 4. Other examples of antibodies which inhibit HERdimerization include antibodies which bind to EGFR and inhibitdimerization thereof with one or more other HER receptors (for exampleEGFR monoclonal antibody 806, MAb 806, which binds to activated or“untethered” EGFR; see Johns et al., J. Biol. Chem. 279(29):30375-30384(2004)); antibodies which bind to HER3 and inhibit dimerization thereofwith one or more other HER receptors; and antibodies which bind to HER4and inhibit dimerization thereof with one or more other HER receptors.

An antibody which “inhibits HER dimerization more effectively thantrastuzumab” is one which reduces or eliminates HER dimers moreeffectively (for example at least about 2-fold more effectively) thantrastuzumab. Preferably, such an antibody inhibits HER2 dimerization atleast about as effectively as an antibody selected from the groupconsisting of murine monoclonal antibody 2C4, a Fab fragment of murinemonoclonal antibody 2C4, pertuzumab, and a Fab fragment of pertuzumab.One can evaluate HER dimerization inhibition by studying HER dimersdirectly, or by evaluating HER activation, or downstream signaling,which results from HER dimerization, and/or by evaluating theantibody-HER2 binding site, etc. Assays for screening for antibodieswith the ability to inhibit HER dimerization more effectively thantrastuzumab are described in Agus et al. Cancer Cell 2: 127-137 (2002)and WO01/00245 (Adams et al.). By way of example only, one may assay forinhibition of HER dimerization by assessing, for example, inhibition ofHER dimer formation (see, e.g., FIG. 1A-B of Agus et al. Cancer Cell 2:127-137 (2002); and WO01/00245); reduction in HER ligand activation ofcells which express HER dimers (WO01/00245 and FIG. 2A-B of Agus et al.Cancer Cell 2: 127-137 (2002), for example); blocking of HER ligandbinding to cells which express HER dimers (WO01/00245, and FIG. 2E ofAgus et al. Cancer Cell 2: 127-137 (2002), for example); cell growthinhibition of cancer cells (e.g. MCF7, MDA-MD-134, ZR-75-1, MD-MB-175,T-47D cells) which express HER dimers in the presence (or absence) ofHER ligand (WO01/00245 and FIGS. 3A-D of Agus et al. Cancer Cell 2:127-137 (2002), for instance); inhibition of downstream signaling (forinstance, inhibition of HRG-dependent AKT phosphorylation or inhibitionof HRG- or TGFα-dependent MAPK phosphorylation) (see, WO01/00245, andFIG. 2C-D of Agus et al. Cancer Cell 2: 127-137 (2002), for example).One may also assess whether the antibody inhibits HER dimerization bystudying the antibody-HER2 binding site, for instance, by evaluating astructure or model, such as a crystal structure, of the antibody boundto HER2 (See, for example, Franklin et al. Cancer Cell 5:317-328(2004)).

A “heterodimeric binding site” on HER2, refers to a region in theextracellular domain of HER2 that contacts, or interfaces with, a regionin the extracellular domain of EGFR, HER3 or HER4 upon formation of adimer therewith. The region is found in Domain II of HER2. Franklin etal. Cancer Cell 5:317-328 (2004).

The HER2 antibody may “inhibit HRG-dependent AKT phosphorylation” and/orinhibit “HRG- or TGFα-dependent MAPK phosphorylation” more effectively(for instance at least 2-fold more effectively) than trastuzumab (seeAgus et al. Cancer Cell 2: 127-137 (2002) and WO01/00245, by way ofexample).

The HER2 antibody may be one which does “not inhibit HER2 ectodomaincleavage” (Molina et al. Cancer Res. 61:4744-4749 (2001)).

A HER2 antibody that “binds to a heterodimeric binding site” of HER2,binds to residues in domain II (and optionally also binds to residues inother of the domains of the HER2 extracellular domain, such as domains Iand III), and can sterically hinder, at least to some extent, formationof a HER2-EGFR, HER2-HER3, or HER2-HER4 heterodimer. Franklin et al.Cancer Cell 5:317-328 (2004) characterize the HER2-pertuzumab crystalstructure, deposited with the RCSB Protein Data Bank (ID Code IS78),illustrating an exemplary antibody that binds to the heterodimericbinding site of HER2.

An antibody that “binds to domain II” of HER2 binds to residues indomain II and optionally residues other domain(s) of HER2, such asdomains I and III. Preferably the antibody that binds to domain II bindsto the junction between domains I, II and III of HER2.

A “native sequence” polypeptide is one which has the same amino acidsequence as a polypeptide (e.g., HER receptor or HER ligand) derivedfrom nature. Such native sequence polypeptides can be isolated fromnature or can be produced by recombinant or synthetic means. Thus, anative sequence polypeptide can have the amino acid sequence ofnaturally occurring human polypeptide, murine polypeptide, orpolypeptide from any other mammalian species.

The term “antibody” herein is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments, so long as theyexhibit the desired biological activity.

The term “monoclonal antibody” as used herein refers to an antibody froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical and/orbind the same epitope(s), except for possible variants that may ariseduring production of the monoclonal antibody, such variants generallybeing present in minor amounts. Such monoclonal antibody typicallyincludes an antibody comprising a polypeptide sequence that binds atarget, wherein the target-binding polypeptide sequence was obtained bya process that includes the selection of a single target bindingpolypeptide sequence from a plurality of polypeptide sequences. Forexample, the selection process can be the selection of a unique clonefrom a plurality of clones, such as a pool of hybridoma clones, phageclones or recombinant DNA clones. It should be understood that theselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity, themonoclonal antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by a variety of techniques,including, for example, the hybridoma method (e.g., Kohler et al.,Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual,(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al.,in: Monoclonal Antibodies and T-Cell Hybridomas 563-681, (Elsevier,N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No.4,816,567), phage display technologies (see, e.g., Clackson et al.,Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol., 222:581-597(1991); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al.,J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci.USA 101(34):12467-12472 (2004); and Lee et al. J. Immunol. Methods284(1-2):119-132 (2004), and technologies for producing human orhuman-like antibodies in animals that have parts or all of the humanimmunoglobulin loci or genes encoding human immunoglobulin sequences(see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741;Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Yearin Immuno., 7:33 (1993); U.S. Pat. Nos. 5,545,806; 5,569,825; 5,591,669(all of GenPharm); U.S. Pat. No. 5,545,807; WO 1997/17852; U.S. Pat.Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and5,661,016; Marks et al., Bio/Technology, 10: 779-783 (1992); Lonberg etal., Nature, 368: 856-859 (1994); Morrison, Nature, 368: 812-813 (1994);Fishwild et al., Nature Biotechnology, 14: 845-851 (1996); Neuberger,Nature Biotechnology, 14: 826 (1996); and Lonberg and Huszar, Intern.Rev. Immunol., 13: 65-93 (1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimericantibodies of interest herein include “primatized” antibodies comprisingvariable domain antigen-binding sequences derived from a non-humanprimate (e.g. Old World Monkey, Ape etc) and human constant regionsequences.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen binding region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragment(s).

An “intact antibody” herein is one which comprises two antigen bindingregions, and an Fc region. Preferably, the intact antibody has one ormore effector functions.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes”.There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called α, δ, ε, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

Antibody “effector functions” refer to those biological activitiesattributable to an Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; down regulation of cell surfacereceptors (e.g. B cell receptor; BCR), etc.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells in summarized is Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Toassess ADCC activity of a molecule of interest, an in vitro ADCC assay,such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source thereof, e.g. from blood or PBMCs asdescribed herein.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. Activating receptor FcγRIIA contains animmunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmicdomain. Inhibiting receptor FcγRIIB contains an immunoreceptortyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (seereview M. in Daëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs arereviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capelet al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.Med. 126:330-41 (1995). Other FcRs, including those to be identified inthe future, are encompassed by the term “FcR” herein. The term alsoincludes the neonatal receptor, FcRn, which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), and regulateshomeostasis of immunoglobulins.

“Complement dependent cytotoxicity” or “CDC” refers to the ability of amolecule to lyse a target in the presence of complement. The complementactivation pathway is initiated by the binding of the first component ofthe complement system (C1q) to a molecule (e.g. an antibody) complexedwith a cognate antigen. To assess complement activation, a CDC assay,e.g. as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163(1996), may be performed.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend. The constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light-chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework regions (FRs). The variabledomains of native heavy and light chains each comprise four FRs, largelyadopting a 3-sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the 3-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (e.g. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “FrameworkRegion” or “FR” residues are those variable domain residues other thanthe hypervariable region residues as herein defined.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-binding sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear at least one free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The “light chains” of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (κ) andlambda (λ), based on the amino acid sequences of their constant domains.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thescFv to form the desired structure for antigen binding. For a review ofscFv see Plückthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994). HER2 antibody scFv fragments are described in WO93/16185; U.S.Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a variable heavy domain(V_(H)) connected to a variable light domain (V_(L)) in the samepolypeptide chain (V_(H)-V_(L)). By using a linker that is too short toallow pairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

Humanized HER2 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3,huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 ortrastuzumab (HERCEPTIN®) as described in Table 3 of U.S. Pat. No.5,821,337 expressly incorporated herein by reference; humanized 520C9(WO93/21319); and humanized 2C4 antibodies as described herein.

For the purposes herein, “trastuzumab,” “HERCEPTIN®,” and “huMAb4D5-8”refer to an antibody comprising the light and heavy chain amino acidsequences in SEQ ID NOS. 15 and 16, respectively.

Herein, “pertuzumab” and “OMNITARG™” refer to an antibody comprising thelight and heavy chain amino acid sequences in SEQ ID NOS. 13 and 14,respectively.

Differences between trastuzumab and pertuzumab functions are illustratedin FIG. 6.

A “naked antibody” is an antibody that is not conjugated to aheterologous molecule, such as a cytotoxic moiety or radiolabel.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An “affinity matured” antibody is one with one or more alterations inone or more hypervariable regions thereof which result an improvement inthe affinity of the antibody for antigen, compared to a parent antibodywhich does not possess those alteration(s). Preferred affinity maturedantibodies will have nanomolar or even picomolar affinities for thetarget antigen Affinity matured antibodies are produced by proceduresknown in the art. Marks et al. Bio/Technology 10:779-783 (1992)describes affinity maturation by VH and VL domain shuffling. Randommutagenesis of CDR and/or framework residues is described by: Barbas etal. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995);Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J.Mol. Biol. 226:889-896 (1992).

The term “main species antibody” herein refers to the antibody structurein a composition which is the quantitatively predominant antibodymolecule in the composition. In one embodiment, the main speciesantibody is a HER2 antibody, such as an antibody that binds to Domain IIof HER2, antibody that inhibits HER dimerization more effectively thantrastuzumab, and/or an antibody which binds to a heterodimeric bindingsite of HER2. The preferred embodiment herein of the main speciesantibody is one comprising the variable light and variable heavy aminoacid sequences in SEQ ID Nos. 3 and 4, and most preferably comprisingthe light chain and heavy chain amino acid sequences in SEQ ID Nos. 13and 14 (pertuzumab).

An “amino acid sequence variant” antibody herein is an antibody with anamino acid sequence which differs from a main species antibody.Ordinarily, amino acid sequence variants will possess at least about 70%homology with the main species antibody, and preferably, they will be atleast about 80%, more preferably at least about 90% homologous with themain species antibody. The amino acid sequence variants possesssubstitutions, deletions, and/or additions at certain positions withinor adjacent to the amino acid sequence of the main species antibody.Examples of amino acid sequence variants herein include acidic variant(e.g. deamidated antibody variant), basic variant, the antibody with anamino-terminal leader extension (e.g. VHS-) on one or two light chainsthereof, antibody with a C-terminal lysine residue on one or two heavychains thereof, etc, and includes combinations of variations to theamino acid sequences of heavy and/or light chains. The antibody variantof particular interest herein is the antibody comprising anamino-terminal leader extension on one or two light chains thereof,optionally further comprising other amino acid sequence and/orglycosylation differences relative to the main species antibody.

A “glycosylation variant” antibody herein is an antibody with one ormore carbohydrate moeities attached thereto which differ from one ormore carbohydate moieties attached to a main species antibody. Examplesof glycosylation variants herein include antibody with a G1 or G2oligosaccharide structure, instead a G0 oligosaccharide structure,attached to an Fc region thereof, antibody with one or two carbohydratemoieties attached to one or two light chains thereof, antibody with nocarbohydrate attached to one or two heavy chains of the antibody, etc,and combinations of glycosylation alterations.

Where the antibody has an Fc region, an oligosaccharide structure suchas that shown in FIG. 9 herein may be attached to one or two heavychains of the antibody, e.g. at residue 299 (298, Eu numbering ofresidues). For pertuzumab, G0 was the predominant oligosaccharidestructure, with other oligosaccharide structures such as G0-F, G-1,Man5, Man6, G1-1, G1(1-6), G1(1-3) and G2 being found in lesser amountsin the pertuzumab composition.

Unless indicated otherwise, a “G1 oligosaccharide structure” hereinincludes G-1, G1-1, G1(1-6) and G1(1-3) structures.

An “amino-terminal leader extension” herein refers to one or more aminoacid residues of the amino-terminal leader sequence that are present atthe amino-terminus of any one or more heavy or light chains of anantibody. An exemplary amino-terminal leader extension comprises orconsists of three amino acid residues, VHS, present on one or both lightchains of an antibody variant.

A “deamidated” antibody is one in which one or more asparagine residuesthereof has been derivitized, e.g. to an aspartic acid, a succinimide,or an iso-aspartic acid.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include, but are not limitedto, carcinoma, lymphoma, blastoma (including medulloblastoma andretinoblastoma), sarcoma (including liposarcoma and synovial cellsarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma,and islet cell cancer), mesothelioma, schwannoma (including acousticneuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include squamouscell cancer (e.g. epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer (SCLC), non-small cell lung cancer(NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung,cancer of the peritoneum, hepatocellular cancer, gastric or stomachcancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer (including metastatic breast cancer),colon cancer, rectal cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, prostatecancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, testicular cancer, esophagael cancer,tumors of the biliary tract, as well as head and neck cancer.

Herein, a “patient” is a human patient. The patient may be a “cancerpatient,” i.e. one who is suffering or at risk for suffering from one ormore symptoms of cancer, or other patient who could benefit from therapywith a HER antibody.

A “biological sample” refers to a sample, generally cells or tissuederived from a biological source.

A “patient sample” refers to a sample obtained from a patient, such as acancer patient.

A “tumor sample” herein is a sample derived from, or comprising tumorcells from, a patient's tumor. Examples of tumor samples herein include,but are not limited to, tumor biopsies, circulating tumor cells,circulating plasma proteins, ascitic fluid, primary cell cultures orcell lines derived from tumors or exhibiting tumor-like properties, aswell as preserved tumor samples, such as formalin-fixed,paraffin-embedded tumor samples or frozen tumor samples.

A “fixed” tumor sample is one which has been histologically preservedusing a fixative.

A “formalin-fixed” tumor sample is one which has been preserved usingformaldehyde as the fixative.

An “embedded” tumor sample is one surrounded by a firm and generallyhard medium such as paraffin, wax, celloidin, or a resin. Embeddingmakes possible the cutting of thin sections for microscopic examinationor for generation of tissue microarrays (TMAs).

A “paraffin-embedded” tumor sample is one surrounded by a purifiedmixture of solid hydrocarbons derived from petroleum.

Herein, a “frozen” tumor sample refers to a tumor sample which is, orhas been, frozen.

A cancer or biological sample which “displays HER expression,amplification, or activation” is one which, in a diagnostic test,expresses (including overexpresses) a HER receptor, has amplified HERgene, and/or otherwise demonstrates activation or phosphorylation of aHER receptor. Such activation can be determined directly (e.g. bymeasuring HER phosphorylation) or indirectly (e.g. by gene expressionprofiling or by detecting HER heterodimers, as described herein).

A cancer with “HER receptor overexpression or amplification” is onewhich has significantly higher levels of a HER receptor protein or genecompared to a noncancerous cell of the same tissue type. Suchoverexpression may be caused by gene amplification or by increasedtranscription or translation. HER receptor overexpression oramplification may be determined in a diagnostic or prognostic assay byevaluating increased levels of the HER protein present on the surface ofa cell (e.g. via an immunohistochemistry assay; IHC). Alternatively, oradditionally, one may measure levels of HER-encoding nucleic acid in thecell, e.g. via fluorescent in situ hybridization (FISH; see WO98/45479published October, 1998), southern blotting, or polymerase chainreaction (PCR) techniques, such as quantitative real time PCR (qRT-PCR).One may also study HER receptor overexpression or amplification bymeasuring shed antigen (e.g., HER extracellular domain) in a biologicalfluid such as serum (see, e.g., U.S. Pat. No. 4,933,294 issued Jun. 12,1990; WO91/05264 published Apr. 18, 1991; U.S. Pat. No. 5,401,638 issuedMar. 28, 1995; and Sias et al. J. Immunol. Methods 132: 73-80 (1990)).Aside from the above assays, various in vivo assays are available to theskilled practitioner. For example, one may expose cells within the bodyof the patient to an antibody which is optionally labeled with adetectable label, e.g. a radioactive isotope, and binding of theantibody to cells in the patient can be evaluated, e.g. by externalscanning for radioactivity or by analyzing a biopsy taken from a patientpreviously exposed to the antibody.

Conversely, a cancer which “does not overexpress or amplify HERreceptor” is one which does not have higher than normal levels of HERreceptor protein or gene compared to a noncancerous cell of the sametissue type. Antibodies that inhibit HER dimerization, such aspertuzumab, may be used to treat cancer which does not overexpress oramplify HER2 receptor.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially a HER expressingcancer cell either in vitro or in vivo. Thus, the growth inhibitoryagent may be one which significantly reduces the percentage of HERexpressing cells in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topo II inhibitors such as doxorubicin,epirubicin, daunorubicin, etoposide, and bleomycin. Those agents thatarrest G1 also spill over into S-phase arrest, for example, DNAalkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13.

Examples of “growth inhibitory” antibodies are those which bind to HER2and inhibit the growth of cancer cells overexpressing HER2. Preferredgrowth inhibitory HER2 antibodies inhibit growth of SK-BR-3 breast tumorcells in cell culture by greater than 20%, and preferably greater than50% (e.g. from about 50% to about 100%) at an antibody concentration ofabout 0.5 to 30 μg/ml, where the growth inhibition is determined sixdays after exposure of the SK-BR-3 cells to the antibody (see U.S. Pat.No. 5,677,171 issued Oct. 14, 1997). The SK-BR-3 cell growth inhibitionassay is described in more detail in that patent and hereinbelow. Thepreferred growth inhibitory antibody is a humanized variant of murinemonoclonal antibody 4D5, e.g., trastuzumab.

An antibody which “induces apoptosis” is one which induces programmedcell death as determined by binding of annexin V, fragmentation of DNA,cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation,and/or formation of membrane vesicles (called apoptotic bodies). Thecell is usually one which overexpresses the HER2 receptor. Preferablythe cell is a tumor cell, e.g. a breast, ovarian, stomach, endometrial,salivary gland, lung, kidney, colon, thyroid, pancreatic or bladdercell. In vitro, the cell may be a SK-BR-3, BT474, Calu 3 cell,MDA-MB-453, MDA-MB-361 or SKOV3 cell. Various methods are available forevaluating the cellular events associated with apoptosis. For example,phosphatidyl serine (PS) translocation can be measured by annexinbinding; DNA fragmentation can be evaluated through DNA laddering; andnuclear/chromatin condensation along with DNA fragmentation can beevaluated by any increase in hypodiploid cells. Preferably, the antibodywhich induces apoptosis is one which results in about 2 to 50 fold,preferably about 5 to 50 fold, and most preferably about 10 to 50 fold,induction of annexin binding relative to untreated cell in an annexinbinding assay using BT474 cells (see below). Examples of HER2 antibodiesthat induce apoptosis are 7C2 and 7F3.

The “epitope 2C4” is the region in the extracellular domain of HER2 towhich the antibody 2C4 binds. In order to screen for antibodies whichbind to the 2C4 epitope, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Preferably the antibody blocks 2C4's binding to HER2 by about 50% ormore. Alternatively, epitope mapping can be performed to assess whetherthe antibody binds to the 2C4 epitope of HER2. Epitope 2C4 comprisesresidues from Domain II in the extracellular domain of HER2. 2C4 andpertuzumab binds to the extracellular domain of HER2 at the junction ofdomains I, II and III. Franklin et al. Cancer Cell 5:317-328 (2004).

The “epitope 4D5” is the region in the extracellular domain of HER2 towhich the antibody 4D5 (ATCC CRL 10463) and trastuzumab bind. Thisepitope is close to the transmembrane domain of HER2, and within DomainIV of HER2. To screen for antibodies which bind to the 4D5 epitope, aroutine cross-blocking assay such as that described in Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and DavidLane (1988), can be performed. Alternatively, epitope mapping can beperformed to assess whether the antibody binds to the 4D5 epitope ofHER2 (e.g. any one or more residues in the region from about residue 529to about residue 625, inclusive of the HER2 ECD, residue numberingincluding signal peptide).

The “epitope 7C2/7F3” is the region at the N terminus, within Domain I,of the extracellular domain of HER2 to which the 7C2 and/or 7F3antibodies (each deposited with the ATCC, see below) bind. To screen forantibodies which bind to the 7C2/7F3 epitope, a routine cross-blockingassay such as that described in Antibodies, A Laboratory Manual, ColdSpring Harbor Laboratory, Ed Harlow and David Lane (1988), can beperformed. Alternatively, epitope mapping can be performed to establishwhether the antibody binds to the 7C2/7F3 epitope on HER2 (e.g. any oneor more of residues in the region from about residue 22 to about residue53 of the HER2 ECD, residue numbering including signal peptide).

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disease as well as those in which the disease is to beprevented. Hence, the patient to be treated herein may have beendiagnosed as having the disease or may be predisposed or susceptible tothe disease.

The term “effective amount” refers to an amount of a drug effective totreat cancer in the patient. The effective amount of the drug may reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., slowto some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. The effectiveamount may extend progression free survival (e.g. as measured byResponse Evaluation Criteria for Solid Tumors, RECIST, or CA-125changes), result in an objective response (including a partial response,PR, or complete respose, CR), increase overall survival time, and/orimprove one or more symptoms of cancer (e.g. as assessed by FOSI).

“Overall survival” refers to the patient remaining alive for a definedperiod of time, such as 1 year, 5 years, etc, e.g., from the time ofdiagnosis or treatment.

“Progression free survival” refers to the patient remaining alive,without the cancer getting worse. An “objective response” refers to ameasurable response, including complete response (CR) or partialresponse (PR).

By “complete response” or “complete remission” is intended thedisappearance of all signs of cancer in response to treatment. This doesnot always mean the cancer has been cured.

“Partial response” refers to a decrease in the size of one or moretumors or lesions, or in the extent of cancer in the body, in responseto treatment.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; TLK 286 (TELCYTA™); acetogenins (especiallybullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol,MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; acamptothecin (including the synthetic analogue topotecan (HYCAMTIN®),CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;bisphosphonates, such as clodronate; antibiotics such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin gamma1I andcalicheamicin omegaI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)) and anthracyclines such as annamycin, AD 32,alcarubicin, daunorubicin, dexrazoxane, DX-52-1, epirubicin, GPX-100,idarubicin, KRN5500, menogaril, dynemicin, including dynemicin A, anesperamicin, neocarzinostatin chromophore and related chromoproteinenediyne antiobiotic chromophores, aclacinomysins, actinomycin,authramycin, azaserine, bleomycins, cactinomycin, carabicin,caminomycin, carzinophilin, chromomycinis, dactinomycin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin, liposomal doxorubicin, and deoxydoxorubicin),esorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin; folic acid analogues such asdenopterin, pteropterin, and trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, and testolactone; anti-adrenals such as aminoglutethimide,mitotane, and trilostane; folic acid replenisher such as folinic acid(leucovorin); aceglatone; anti-folate anti-neoplastic agents such asALIMTA®, LY231514 pemetrexed, dihydrofolate reductase inhibitors such asmethotrexate, anti-metabolites such as 5-fluorouracil (5-FU) and itsprodrugs such as UFT, S-1 and capecitabine, and thymidylate synthaseinhibitors and glycinamide ribonucleotide formyltransferase inhibitorssuch as raltitrexed (TOMUDEX®, TDX); inhibitors of dihydropyrimidinedehydrogenase such as eniluracil; aldophosphamide glycoside;aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate;defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate;an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;lonidainine; maytansinoids such as maytansine and ansamitocins;mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin;phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine;PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.);razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine(ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol;mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”);cyclophosphamide; thiotepa; taxoids and taxanes, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® docetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; platinum; platinumanalogs or platinum-based analogs such as cisplatin, oxaliplatin andcarboplatin; vinblastine (VELBAN®); etoposide (VP-16); ifosfamide;mitoxantrone; vincristine (ONCOVIN®); vinca alkaloid; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; xeloda;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; pharmaceutically acceptablesalts, acids or derivatives of any of the above; as well as combinationsof two or more of the above such as CHOP, an abbreviation for a combinedtherapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone,and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin(ELOXATIN™) combined with 5-FU and leucovorin.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON® toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those thatinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, andepidermal growth factor receptor (EGF-R); vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELIX® rmRH; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

An “antimetabolite chemotherapeutic agent” is an agent which isstructurally similar to a metabolite, but can not be used by the body ina productive manner. Many antimetabolite chemotherapeutic agentsinterfere with the production of the nucleic acids, RNA and DNA.Examples of antimetabolite chemotherapeutic agents include gemcitabine(GEMZAR®), 5-fluorouracil (5-FU), capecitabine (XELODA™),6-mercaptopurine, methotrexate, 6-thioguanine, pemetrexed, raltitrexed,arabinosylcytosine ARA-C cytarabine (CYTOSAR-U®), dacarbazine(DTIC-DOME®), azocytosine, deoxycytosine, pyridmidene, fludarabine(FLUDARA®), cladrabine, 2-deoxy-D-glucose etc. The preferredantimetabolite chemotherapeutic agent is gemcitabine.

“Gemcitabine” or “2′-deoxy-2′,2′-difluorocytidine monohydrochloride(b-isomer)” is a nucleoside analogue that exhibits antitumor activity.The empirical formula for gemcitabine HCl is C9H11F2N3O4.HCl.Gemcitabine HCl is sold by Eli Lilly under the trademark GEMZAR®.

A “platinum-based chemotherapeutic agent” comprises an organic compoundwhich contains platinum as an integral part of the molecule. Examples ofplatinum-based chemotherapeutic agents include carboplatin, cisplatin,and oxaliplatinum.

By “platinum-based chemotherapy” is intended therapy with one or moreplatinum-based chemotherapeutic agents, optionally in combination withone or more other chemotherapeutic agents.

By “platinum resistant” cancer is meant that the cancer patient hasprogressed while receiving platinum-based chemotherapy (i.e. the patientis “platinum refractory”), or the patient has progressed within 12months (for instance, within 6 months) after completing a platinum-basedchemotherapy regimen.

An “anti-angiogenic agent” refers to a compound which blocks, orinterferes with to some degree, the development of blood vessels. Theanti-angiogenic factor may, for instance, be a small molecule orantibody that binds to a growth factor or growth factor receptorinvolved in promoting angiogenesis. The preferred anti-angiogenic factorherein is an antibody that binds to vascular endothelial growth factor(VEGF), such as bevacizumab (AVASTIN®).

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis factor such asTNF-α or TNF-β; and other polypeptide factors including LIF and kitligand (KL). As used herein, the term cytokine includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

As used herein, the term “EGFR-targeted drug” refers to a therapeuticagent that binds to EGFR and, optionally, inhibits EGFR activation.Examples of such agents include antibodies and small molecules that bindto EGFR. Examples of antibodies which bind to EGFR include MAb 579 (ATCCCRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb528 (ATCC CRL 8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn et al.)and variants thereof, such as chimerized 225 (C225 or Cetuximab;ERBUTIX®) and reshaped human 225 (H225) (see, WO 96/40210, ImcloneSystems Inc.); antibodies that bind type II mutant EGFR (U.S. Pat. No.5,212,290); humanized and chimeric antibodies that bind EGFR asdescribed in U.S. Pat. No. 5,891,996; and human antibodies that bindEGFR, such as ABX-EGF (see WO98/50433, Abgenix); EMD 55900 (Stragliottoet al. Eur. J. Cancer 32A:636-640 (1996)); and mAb 806 or humanized mAb806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). Theanti-EGFR antibody may be conjugated with a cytotoxic agent, thusgenerating an immunoconjugate (see, e.g., EP659,439A2, Merck PatentGmbH). Examples of small molecules that bind to EGFR include ZD1839 orGefitinib (IRESSA™; Astra Zeneca); CP-358774 or Erlotinib HCL (TARCEVA™;Genentech/OSI); and AG1478, AG1571 (SU 5271; Sugen).

A “tyrosine kinase inhibitor” is a molecule which inhibits tyrosinekinase activity of a tyrosine kinase such as a HER receptor. Examples ofsuch inhibitors include the EGFR-targeted drugs noted in the precedingparagraph; small molecule HER2 tyrosine kinase inhibitor such as TAK165available from Takeda; dual-HER inhibitors such as EKB-569 (availablefrom Wyeth) which preferentially binds EGFR but inhibits both HER2 andEGFR-overexpressing cells; GW572016 (available from Glaxo) an oral HER2and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis);pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1inhibitors such as antisense agent ISIS-5132 available from ISISPharmaceuticals which inhibits Raf-1 signaling; non-HER targeted TKinhibitors such as Imatinib mesylate (Gleevac™) available from Glaxo;MAPK extracellular regulated kinase I inhibitor CI-1040 (available fromPharmacia); quinazolines, such as PD 153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines,such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines,4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostines containingnitrothiophene moieties; PD-0183805 (Warner-Lamber); antisense molecules(e.g. those that bind to HER-encoding nucleic acid); quinoxalines (U.S.Pat. No. 5,804,396); tryphostins (U.S. Pat. No. 5,804,396); ZD6474(Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors suchas CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly); Imatinibmesylate (Gleevac; Novartis); PKI 166 (Novartis); GW2016 (GlaxoSmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Sugen);ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11(Imclone); or as described in any of the following patent publications:U.S. Pat. No. 5,804,396; WO99/09016 (American Cyanimid); WO98/43960(American Cyanamid); WO97/38983 (Warner Lambert); WO99/06378 (WarnerLambert); WO99/06396 (Warner Lambert); WO96/30347 (Pfizer, Inc);WO96/33978 (Zeneca); WO96/3397 (Zeneca); and WO96/33980 (Zeneca).

An “autoimmune disease” herein is a disease or disorder arising from anddirected against an individual's own tissues or a co-segregate ormanifestation thereof or resulting condition therefrom. Examples ofautoimmune diseases or disorders include, but are not limited toarthritis (rheumatoid arthritis such as acute arthritis, chronicrheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronicinflammatory arthritis, degenerative arthritis, infectious arthritis,Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebralarthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis,arthritis chronica progrediente, arthritis deformans, polyarthritischronica primaria, reactive arthritis, and ankylosing spondylitis),inflammatory hyperproliferative skin diseases, psoriasis such as plaquepsoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of thenails, atopy including atopic diseases such as hay fever and Job'ssyndrome, dermatitis including contact dermatitis, chronic contactdermatitis, allergic dermatitis, allergic contact dermatitis, dermatitisherpetiformis, and atopic dermatitis, x-linked hyper IgM syndrome,urticaria such as chronic allergic urticaria and chronic idiopathicurticaria, including chronic autoimmune urticaria,polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermalnecrolysis, scleroderma (including systemic scleroderma), sclerosis suchas systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS,primary progressive MS (PPMS), and relapsing remitting MS (RRMS),progressive systemic sclerosis, atherosclerosis, arteriosclerosis,sclerosis disseminata, and ataxic sclerosis, inflammatory bowel disease(IBD) (for example, Crohn's disease, autoimmune-mediatedgastrointestinal diseases, colitis such as ulcerative colitis, colitisulcerosa, microscopic colitis, collagenous colitis, colitis polyposa,necrotizing enterocolitis, and transmural colitis, and autoimmuneinflammatory bowel disease), pyoderma gangrenosum, erythema nodosum,primary sclerosing cholangitis, episcleritis), respiratory distresssyndrome, including adult or acute respiratory distress syndrome (ARDS),meningitis, inflammation of all or part of the uvea, iritis,choroiditis, an autoimmune hematological disorder, rheumatoidspondylitis, sudden hearing loss, IgE-mediated diseases such asanaphylaxis and allergic and atopic rhinitis, encephalitis such asRasmussen's encephalitis and limbic and/or brainstem encephalitis,uveitis, such as anterior uveitis, acute anterior uveitis, granulomatousuveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterioruveitis, or autoimmune uveitis, glomerulonephritis (GN) with and withoutnephrotic syndrome such as chronic or acute glomerulonephritis such asprimary GN, immune-mediated GN, membranous GN (membranous nephropathy),idiopathic membranous GN or idiopathic membranous nephropathy, membrano-or membranous proliferative GN (MPGN), including Type I and Type II, andrapidly progressive GN, allergic conditions and responses, allergicreaction, eczema including allergic or atopic eczema, asthma such asasthma bronchiale, bronchial asthma, and auto-immune asthma, conditionsinvolving infiltration of T cells and chronic inflammatory responses,immune reactions against foreign antigens such as fetal A-B-O bloodgroups during pregnancy, chronic pulmonary inflammatory disease,autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupuserythematosus (SLE) or systemic lupus erythematodes such as cutaneousSLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome(NLE), lupus erythematosus disseminatus, lupus (including nephritis,cerebritis, pediatric, non-renal, extra-renal, discoid, alopecia),juvenile onset (Type I) diabetes mellitus, including pediatricinsulin-dependent diabetes mellitus (IDDM), adult onset diabetesmellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetesinsipidus, immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis,sarcoidosis, granulomatosis including lymphomatoid granulomatosis,Wegener's granulomatosis, agranulocytosis, vasculitides, includingvasculitis (including large vessel vasculitis (including polymyalgiarheumatica and giant cell (Takayasu's) arteritis), medium vesselvasculitis (including Kawasaki's disease and polyarteritisnodosa/periarteritis nodosa), microscopic polyarteritis, CNS vasculitis,necrotizing, cutaneous, or hypersensitivity vasculitis, systemicnecrotizing vasculitis, and ANCA-associated vasculitis, such asChurg-Strauss vasculitis or syndrome (CSS)), temporal arteritis,aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia,Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemiaincluding autoimmune hemolytic anemia (AIHA), pernicious anemia (anemiaperniciosa), Addison's disease, pure red cell anemia or aplasia (PRCA),Factor VIII deficiency, hemophilia A, autoimmune neutropenia,pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNSinflammatory disorders, multiple organ injury syndrome such as thosesecondary to septicemia, trauma or hemorrhage, antigen-antibodycomplex-mediated diseases, anti-glomerular basement membrane disease,anti-phospholipid antibody syndrome, allergic neuritis, Bechet's orBehcet's disease, Castleman's syndrome, Goodpasture's syndrome,Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome,pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus(including pemphigus vulgaris, pemphigus foliaceus, pemphigusmucus-membrane pemphigoid, and pemphigus erythematosus), autoimmunepolyendocrinopathies, Reiter's disease or syndrome, immune complexnephritis, antibody-mediated nephritis, neuromyelitis optica,polyneuropathies, chronic neuropathy such as IgM polyneuropathies orIgM-mediated neuropathy, thrombocytopenia (as developed by myocardialinfarction patients, for example), including thrombotic thrombocytopenicpurpura (TTP), post-transfusion purpura (PTP), heparin-inducedthrombocytopenia, and autoimmune or immune-mediated thrombocytopeniasuch as idiopathic thrombocytopenic purpura (ITP) including chronic oracute ITP, autoimmune disease of the testis and ovary includingautoimmune orchitis and oophoritis, primary hypothyroidism,hypoparathyroidism, autoimmune endocrine diseases including thyroiditissuch as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis(Hashimoto's thyroiditis), or subacute thyroiditis, autoimmune thyroiddisease, idiopathic hypothyroidism, Grave's disease, polyglandularsyndromes such as autoimmune polyglandular syndromes (or polyglandularendocrinopathy syndromes), paraneoplastic syndromes, includingneurologic paraneoplastic syndromes such as Lambert-Eaton myasthenicsyndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome,encephalomyelitis such as allergic encephalomyelitis orencephalomyelitis allergica and experimental allergic encephalomyelitis(EAE), myasthenia gravis such as thymoma-associated myasthenia gravis,cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonusmyoclonus syndrome (OMS), and sensory neuropathy, multifocal motorneuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis,lupoid hepatitis, giant cell hepatitis, chronic active hepatitis orautoimmune chronic active hepatitis, lymphoid interstitial pneumonitis(LIP), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barrésyndrome, Berger's disease (IgA nephropathy), idiopathic IgAnephropathy, linear IgA dermatosis, primary biliary cirrhosis,pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac disease,Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue,idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS;Lou Gehrig's disease), coronary artery disease, autoimmune ear diseasesuch as autoimmune inner ear disease (AIED), autoimmune hearing loss,opsoclonus myoclonus syndrome (OMS), polychondritis such as refractoryor relapsed polychondritis, pulmonary alveolar proteinosis, amyloidosis,scleritis, a non-cancerous lymphocytosis, a primary lymphocytosis, whichincludes monoclonal B cell lymphocytosis (e.g., benign monoclonalgammopathy and monoclonal garnmopathy of undetermined significance,MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathiessuch as epilepsy, migraine, arrhythmia, muscular disorders, deafness,blindness, periodic paralysis, and channelopathies of the CNS, autism,inflammatory myopathy, focal segmental glomerulosclerosis (FSGS),endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmunehepatological disorder, fibromyalgia, multiple endocrine failure,Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia,demyelinating diseases such as autoimmune demyelinating diseases andchronic inflammatory demyelinating polyneuropathy, diabetic nephropathy,Dressler's syndrome, alopecia greata, CREST syndrome (calcinosis,Raynaud's phenomenon, esophageal dysmotility, sclerodactyl), andtelangiectasia), male and female autoimmune infertility, mixedconnective tissue disease, Chagas' disease, rheumatic fever, recurrentabortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome,Cushing's syndrome, bird-fancier's lung, allergic granulomatousangiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitissuch as allergic alveolitis and fibrosing alveolitis, interstitial lungdisease, transfusion reaction, leprosy, malaria, leishmaniasis,kypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter'ssyndrome, Caplan's syndrome, dengue, endocarditis, endomyocardialfibrosis, diffuse interstitial pulmonary fibrosis, interstitial lungfibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cysticfibrosis, endophthalmitis, erythema elevatum et diutinum,erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome,Felty's syndrome, flariasis, cyclitis such as chronic cyclitis,heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch'scyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV)infection, echovirus infection, cardiomyopathy, Alzheimer's disease,parvovirus infection, rubella virus infection, post-vaccinationsyndromes, congenital rubella infection, Epstein-Barr virus infection,mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea,post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis,tabes dorsalis, chorioiditis, giant cell polymyalgia, endocrineophthamopathy, chronic hypersensitivity pneumonitis,keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathicnephritic syndrome, minimal change nephropathy, benign familial andischemia-reperfusion injury, retinal autoimmunity, joint inflammation,bronchitis, chronic obstructive airway disease, silicosis, aphthae,aphthous stomatitis, arteriosclerotic disorders, aspermiogenese,autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren'scontracture, endophthalmia phacoanaphylactica, enteritis allergica,erythema nodosum leprosum, idiopathic facial paralysis, chronic fatiguesyndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearingloss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,leucopenia, mononucleosis infectiosa, traverse myelitis, primaryidiopathic myxedema, nephrosis, ophthalmia symphatica, orchitisgranulomatosa, pancreatitis, polyradiculitis acuta, pyodermagangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,infertility due to antispermatozoan antobodies, non-malignant thymoma,vitiligo, SCID and Epstein-Barr virus-associated diseases, acquiredimmune deficiency syndrome (AIDS), parasitic diseases such asLesihmania, toxic-shock syndrome, food poisoning, conditions involvinginfiltration of T cells, leukocyte-adhesion deficiency, immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes, diseases involving leukocyte diapedesis, multipleorgan injury syndrome, antigen-antibody complex-mediated diseases,antiglomerular basement membrane disease, allergic neuritis, autoimmunepolyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophicgastritis, sympathetic ophthalmia, rheumatic diseases, mixed connectivetissue disease, nephrotic syndrome, insulitis, polyendocrine failure,peripheral neuropathy, autoimmune polyglandular syndrome type I,adult-onset idiopathic hypoparathyroidism (AOIH), alopecia totalis,dilated cardiomyopathy, epidermolisis bullosa acquisita (EBA),hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosingcholangitis, purulent or nonpurulent sinusitis, acute or chronicsinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, aneosinophil-related disorder such as eosinophilia, pulmonary infiltrationeosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chroniceosinophilic pneumonia, tropical pulmonary eosinophilia,bronchopneumonic aspergillosis, aspergilloma, or granulomas containingeosinophils, anaphylaxis, seronegative spondyloarthritides,polyendocrine autoimmune disease, sclerosing cholangitis, sclera,episclera, chronic mucocutaneous candidiasis, Bruton's syndrome,transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome,ataxia telangiectasia, autoimmune disorders associated with collagendisease, rheumatism, neurological disease, lymphadenitis, ischemicre-perfusion disorder, reduction in blood pressure response, vasculardysfunction, antgiectasis, tissue injury, cardiovascular ischemia,hyperalgesia, cerebral ischemia, and disease accompanyingvascularization, allergic hypersensitivity disorders,glomerulonephritides, reperfusion injury, reperfusion injury ofmyocardial or other tissues, dermatoses with acute inflammatorycomponents, acute purulent meningitis or other central nervous systeminflammatory disorders, ocular and orbital inflammatory disorders,granulocyte transfusion-associated syndromes, cytokine-induced toxicity,narcolepsy, acute serious inflammation, chronic intractableinflammation, pyelitis, pneumonocirrhosis, diabetic retinopathy,diabetic large-artery disorder, endarterial hyperplasia, peptic ulcer,valvulitis, and endometriosis.

A “benign hyperproliferative disorder” is meant a state in a patientthat relates to cell proliferation and which is recognized as abnormalby members of the medical community. An abnormal state is characterizedby a level of a property that is statistically different from the levelobserved in organisms not suffering from the disorder. Cellproliferation refers to growth or extension by multiplication of cellsand includes cell division. The rate of cell proliferation may bemeasured by counting the number of cells produced in a given unit oftime. Examples of benign hyperproliferative disorders include psoriasisand polyps.

A “respiratory disease” involves the respiratory system and includeschronic bronchitis, asthma including acute asthma and allergic asthma,cystic fibrosis, bronchiectasis, allergic or other rhinitis orsinusitis, α1-antitrypsin deficiency, coughs, pulmonary emphysema,pulmonary fibrosis or hyper-reactive airways, chronic obstructivepulmonary disease, and chronic obstructive lung disorder.

“Psoriasis” is a condition characterized by the eruption ofcircumscribed, discrete and confluent, reddish, silvery-scaledmaculopapules. Psoriatic lesions generally occur predominantly on theelbows, knees, scalp, and trunk, and microscopically show characteristicparakerotosis and elongation of rete ridges. The term includes thevarious forms of psoriasis, including erythrodermic, pustular,moderate-severe and recalcitrant forms of the disease.

“Endometriosis” refers to the ectopic occurrence of endometrial tissue,frequently forming cysts containing altered blood.

The term “vascular disease or disorder” herein refers to the variousdiseases or disorders which impact the vascular system, including thecardiovascular system. Examples of such diseases includearteriosclerosis, vascular reobstruction, atherosclerosis, postsurgicalvascular stenosis, restenosis, vascular occlusion or carotid obstructivedisease, coronary artery disease, angina, small vessel disease,hypercholesterolemia, hypertension, and conditions involving abnormalproliferation or function of vascular epithelial cells.

The term “stenosis” refers to narrowing or stricture of a hollow passage(e,g, a duct or canal) in the body.

The term “vascular stenosis” refers to occlusion or narrowing of bloodvessels. Vascular stenosis often results from fatty deposit (as in thecase of atherosclerosis) or excessive migration and proliferation ofvascular smooth muscle cells and endothelial cells. Arteries areparticularly susceptible to stenosis. The term “stenosis” as used hereinspecifically includes initial stenosis and restenosis.

The term “restenosis” refers to recurrence of stenosis after treatmentof initial stenosis with apparent success. For example, “restenosis” inthe context of vascular stenosis, refers to the reoccurrence of vascularstenosis after it has been treated with apparent success, e.g. byremoval of fatty deposit by angioplasty (e.g. percutaneous transluminalcoronary angioplasty), direction coronary atherectomy or stent etc. Oneof the contributing factors in restenosis is intimal hyperplasia. Theterm “intimal hyperplasia”, used interchangeably with “neointimalhyperplasia” and “neointima formation”, refers to thickening of theinner most layer of blood vessels, intima, as a consequence of excessiveproliferation and migration of vascular smooth muscle cells andendothelial cells. The various changes taking place during restenosisare often collectively referred to as “vascular wall remodeling.”

The terms “balloon angioplasty” and “percutaneous transluminal coronaryangioplasty” (PTCA) are often used interchangeably, and refer to anon-surgical catheter-based treatment for removal of plaque from thecoronary artery. Stenosis or restenosis often lead to hypertension as aresult of increased resistance to blood flow.

The term “hypertension” refers to abnormally high blood pressure, i.e.beyond the upper value of the normal range.

“Polyps” refers to a mass of tissue that bulges or projects outward orupward from the normal surface level, thereby being macroscopicallyvisible as a hemispheroidal, speroidal, or irregular moundlike structuregrowing from a relatively broad base or a slender stalk. Examplesinclude colon, rectal and nasal polyps.

“Fibroadenoma” references a benign neoplasm derived from glandularepithelium, in which there is a conspicuous stroma of proliferatingfibroblasts and connective tissue elements. This commonly occurs inbreast tissue.

“Asthma” is a condition which results in difficulty in breathing.Bronchial asthma refers to a condition of the lungs in which there iswidespread narrowing of airways, which may be due to contraction (spasm)of smooth muscle, edema of the mucosa, or mucus in the lumen of thebronchi and bronchioles.

“Bronchitis” refers to inflammation of the mucous membrane of thebronchial tubes.

For the purposes herein, a “vial” refers to a container which holds atherapeutic agent. The vial may be sealed by a stopper pierceable by asyringe. Generally, the vial is formed from glass material. Thetherapeutic agent in the vial can be in various states including liquid,lyophilized, frozen etc.

A “package insert” refers to instructions customarily included incommercial packages of therapeutic agents, that contain informationabout the indications, usage, dosage, administration, contraindicationsand/or warnings concerning the use of such therapeutic products.

II. Production of Antibodies

A description follows as to exemplary techniques for the production ofHER antibodies used in accordance with the present invention. The HERantigen to be used for production of antibodies may be, e.g., a solubleform of the extracellular domain of HER or a portion thereof, containingthe desired epitope. Alternatively, cells expressing HER at their cellsurface (e.g. NIH-3T3 cells transformed to overexpress HER2; or acarcinoma cell line such as SK-BR-3 cells, see Stancovski et al. PNAS(USA) 88:8691-8695 (1991)) can be used to generate antibodies. Otherforms of HER receptor useful for generating antibodies will be apparentto those skilled in the art.

(i) Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

(ii) Monoclonal Antibodies

Various methods for making monoclonal antibodies herein are available inthe art. For example, the monoclonal antibodies may be made using thehybridoma method first described by Kohler et al., Nature, 256:495(1975), by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); and Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce antibody protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells.Review articles on recombinant expression in bacteria of DNA encodingthe antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262(1993) and Plückthun, Immunol. Revs., 130:151-188 (1992).

In a further embodiment, monoclonal antibodies or antibody fragments canbe isolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy chain and light chain constant domains in placeof the homologous murine sequences (U.S. Pat. No. 4,816,567; andMorrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

(iii) Humanized Antibodies

Methods for humanizing non-human antibodies have been described in theart. Preferably, a humanized antibody has one or more amino acidresidues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988)), by substituting hypervariable region sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework region derived fromthe consensus sequence of all human antibodies of a particular subgroupof light or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

WO01/00245 describes production of exemplary humanized HER2 antibodieswhich bind HER2 and block ligand activation of a HER receptor. Thehumanized antibody of particular interest herein blocks EGF, TGF-αand/or HRG mediated activation of MAPK essentially as effectively asmurine monoclonal antibody 2C4 (or a Fab fragment thereof) and/or bindsHER2 essentially as effectively as murine monoclonal antibody 2C4 (or aFab fragment thereof). The humanized antibody herein may, for example,comprise nonhuman hypervariable region residues incorporated into ahuman variable heavy domain and may further comprise a framework region(FR) substitution at a position selected from the group consisting of69H, 71H and 73H utilizing the variable domain numbering system setforth in Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md. (1991). In one embodiment, the humanized antibody comprises FRsubstitutions at two or all of positions 69H, 71H and 73H.

An exemplary humanized antibody of interest herein comprises variableheavy domain complementarity determining residues GFTFTDYTMX, where X ispreferrably D or S (SEQ ID NO:7); DVNPNSGGSIYNQRFKG (SEQ ID NO:8);and/or NLGPSFYFDY (SEQ ID NO:9), optionally comprising amino acidmodifications of those CDR residues, e.g. where the modificationsessentially maintain or improve affinity of the antibody. For example,the antibody variant of interest may have from about one to about sevenor about five amino acid substitutions in the above variable heavy CDRsequences. Such antibody variants may be prepared by affinitymaturation, e.g., as described below. The most preferred humanizedantibody comprises the variable heavy domain amino acid sequence in SEQID NO:4.

The humanized antibody may comprise variable light domaincomplementarity determining residues KASQDVSIGVA (SEQ ID NO:10);SASYX¹X²X³, where X¹ is preferably R or L, X² is preferably Y or E, andX³ is preferably T or S (SEQ ID NO:11); and/or QQYYIYPYT (SEQ ID NO:12),e.g. in addition to those variable heavy domain CDR residues in thepreceding paragraph. Such humanized antibodies optionally comprise aminoacid modifications of the above CDR residues, e.g. where themodifications essentially maintain or improve affinity of the antibody.For example, the antibody variant of interest may have from about one toabout seven or about five amino acid substitutions in the above variablelight CDR sequences. Such antibody variants may be prepared by affinitymaturation, e.g., as described below. The most preferred humanizedantibody comprises the variable light domain amino acid sequence in SEQID NO:3.

The present application also contemplates affinity matured antibodieswhich bind HER2 and block ligand activation of a HER receptor. Theparent antibody may be a human antibody or a humanized antibody, e.g.,one comprising the variable light and/or heavy sequences of SEQ ID Nos.3 and 4, respectively (i.e. variant 574). The affinity matured antibodypreferably binds to HER2 receptor with an affinity superior to that ofmurine 2C4 or variant 574 (e.g. from about two or about four fold, toabout 100 fold or about 1000 fold improved affinity, e.g. as assessedusing a HER2-extracellular domain (ECD) ELISA). Exemplary variable heavyCDR residues for substitution include H28, H30, H34, H35, H64, H96, H99,or combinations of two or more (e.g. two, three, four, five, six, orseven of these residues). Examples of variable light CDR residues foralteration include L28, L50, L53, L56, L91, L92, L93, L94, L96, L97 orcombinations of two or more (e.g. two to three, four, five or up toabout ten of these residues).

Various forms of the humanized antibody or affinity matured antibody arecontemplated. For example, the humanized antibody or affinity maturedantibody may be an antibody fragment, such as a Fab, which is optionallyconjugated with one or more cytotoxic agent(s) in order to generate animmunoconjugate. Alternatively, the humanized antibody or affinitymatured antibody may be an intact antibody, such as an intact IgG1antibody. The preferred intact IgG1 antibody comprises the light chainsequence in SEQ ID NO:13 and the heavy chain sequence in SEQ ID NO:14.

(iv) Human Antibodies

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993); and U.S. Pat. Nos. 5,591,669,5,589,369 and 5,545,807.

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson, Kevin S, andChiswell, David J., Current Opinion in Structural Biology 3:564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature, 352:624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J.12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.

As discussed above, human antibodies may also be generated by in vitroactivated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Human HER2 antibodies are described in U.S. Pat. No. 5,772,997 issuedJun. 30, 1998 and WO 97/00271 published Jan. 3, 1997.

(v) Antibody Fragments

Various techniques have been developed for the production of antibodyfragments comprising one or more antigen binding regions. Traditionally,these fragments were derived via proteolytic digestion of intactantibodies (see, e.g., Morimoto et al., Journal of Biochemical andBiophysical Methods 24:107-117 (1992); and Brennan et al., Science,229:81 (1985)). However, these fragments can now be produced directly byrecombinant host cells. For example, the antibody fragments can beisolated from the antibody phage libraries discussed above.Alternatively, Fab′-SH fragments can be directly recovered from E. coliand chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See WO93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458. Theantibody fragment may also be a “linear antibody”, e.g., as described inU.S. Pat. No. 5,641,870 for example. Such linear antibody fragments maybe monospecific or bispecific.

(vi) Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the HER2 protein. Other suchantibodies may combine a HER2 binding site with binding site(s) forEGFR, HER3 and/or HER4. Alternatively, a HER2 arm may be combined withan arm which binds to a triggering molecule on a leukocyte such as aT-cell receptor molecule (e.g. CD2 or CD3), or Fc receptors for IgG(FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as tofocus cellular defense mechanisms to the HER2-expressing cell.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express HER2. These antibodies possess a HER2-binding armand an arm which binds the cytotoxic agent (e.g. saporin,anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

WO 96/16673 describes a bispecific HER2/FcγRIII antibody and U.S. Pat.No. 5,837,234 discloses a bispecific HER2/FcγRI antibody IDM1 (Osidem).A bispecific HER2/Fcα antibody is shown in WO98/02463. U.S. Pat. No.5,821,337 teaches a bispecific HER2/CD3 antibody. MDX-210 is abispecific HER2-FcγRIII Ab.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain of an antibody constant domain. In thismethod, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g.tyrosine or tryptophan). Compensatory “cavities” of identical or similarsize to the large side chain(s) are created on the interface of thesecond antibody molecule by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

(vii) Other Amino Acid Sequence Modifications

Amino acid sequence modification(s) of the antibodies described hereinare contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody.Amino acid sequence variants of the antibody are prepared by introducingappropriate nucleotide changes into the antibody nucleic acid, or bypeptide synthesis. Such modifications include, for example, deletionsfrom, and/or insertions into and/or substitutions of, residues withinthe amino acid sequences of the antibody. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe antibody, such as changing the number or position of glycosylationsites.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells Science,244:1081-1085 (1989). Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includeantibody with an N-terminal methionyl residue or the antibody fused to acytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g. for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin Table 1 under the heading of “preferred substitutions”. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table 1,or as further described below in reference to amino acid classes, may beintroduced and the products screened.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain Amino acids may begrouped according to similarities in the properties of their side chains(in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, WorthPublishers, New York (1975)):

(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp(W), Met (M)

(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn(N), Gln (O)

(3) acidic: Asp (D), Glu (E)

(4) basic: Lys (K), Arg (R), His(H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking Conversely, cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment such as an Fv fragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g. a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g. 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The antibodyvariants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and human HER2. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody are described in US Pat Appl No US 2003/0157108 A1, Presta, L.See also US 2004/0093621 A1 (Kyowa Hakko Kogyo Co., Ltd). Antibodieswith a bisecting N-acetylglucosamine (GlcNAc) in the carbohydrateattached to an Fc region of the antibody are referenced in WO03/011878,Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodieswith at least one galactose residue in the oligosaccharide attached toan Fc region of the antibody are reported in WO97/30087, Patel et al.See, also, WO98/58964 (Raju, S.) and WO99/22764 (Raju, S.) concerningantibodies with altered carbohydrate attached to the Fc region thereof.

It may be desirable to modify the antibody of the invention with respectto effector function, e.g. so as to enhance antigen-dependentcell-mediated cyotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al. Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).

WO00/42072 (Presta, L.) describes antibodies with improved ADCC functionin the presence of human effector cells, where the antibodies compriseamino acid substitutions in the Fc region thereof. Preferably, theantibody with improved ADCC comprises substitutions at positions 298,333, and/or 334 of the Fc region. Preferably the altered Fc region is ahuman IgG1 Fc region comprising or consisting of substitutions at one,two or three of these positions.

Antibodies with altered C1q binding and/or complement dependentcytotoxicity (CDC) are described in WO99/51642, U.S. Pat. No.6,194,551B1, U.S. Pat. No. 6,242,195B1, U.S. Pat. No. 6,528,624B1 andU.S. Pat. No. 6,538,124 (Idusogie et al.). The antibodies comprise anamino acid substitution at one or more of amino acid positions 270, 322,326, 327, 329, 313, 333 and/or 334 of the Fc region thereof.

To increase the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

Antibodies with improved binding to the neonatal Fc receptor (FcRn), andincreased half-lives, are described in WO00/42072 (Presta, L.). Theseantibodies comprise a Fc region with one or more substitutions thereinwhich improve binding of the Fc region to FcRn. For example, the Fcregion may have substitutions at one or more of positions 238, 256, 265,272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378,380, 382, 413, 424 or 434. The preferred Fc region-comprising antibodyvariant with improved FcRn binding comprises amino acid substitutions atone, two or three of positions 307, 380 and 434 of the Fc regionthereof.

Engineered antibodies with three or more (preferably four) functionalantigen binding sites are also contemplated (US Appln No. US2002/0004587A1, Miller et al.).

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

(viii) Screening for Antibodies with the Desired Properties

Techniques for generating antibodies have been described above. One mayfurther select antibodies with certain biological characteristics, asdesired.

To identify an antibody which blocks ligand activation of a HERreceptor, the ability of the antibody to block HER ligand binding tocells expressing the HER receptor (e.g. in conjugation with another HERreceptor with which the HER receptor of interest forms a HERhetero-oligomer) may be determined. For example, cells naturallyexpressing, or transfected to express, HER receptors of the HERhetero-oligomer may be incubated with the antibody and then exposed tolabeled HER ligand. The ability of the antibody to block ligand bindingto the HER receptor in the HER hetero-oligomer may then be evaluated.

For example, inhibition of HRG binding to MCF7 breast tumor cell linesby HER2 antibodies may be performed using monolayer MCF7 cultures on icein a 24-well-plate format essentially as described in WO01/00245. HER2monoclonal antibodies may be added to each well and incubated for 30minutes. ¹²⁵I-labeled rHRGβ1₁₇₇₋₂₂₄ (25 pm) may then be added, and theincubation may be continued for 4 to 16 hours. Dose response curves maybe prepared and an IC₅₀ value may be calculated for the antibody ofinterest. In one embodiment, the antibody which blocks ligand activationof a HER receptor will have an IC₅₀ for inhibiting HRG binding to MCF7cells in this assay of about 50 nM or less, more preferably 10 nM orless. Where the antibody is an antibody fragment such as a Fab fragment,the IC₅₀ for inhibiting HRG binding to MCF7 cells in this assay may, forexample, be about 100 nM or less, more preferably 50 nM or less.

Alternatively, or additionally, the ability of an antibody to block HERligand-stimulated tyrosine phosphorylation of a HER receptor present ina HER hetero-oligomer may be assessed. For example, cells endogenouslyexpressing the HER receptors or transfected to expressed them may beincubated with the antibody and then assayed for HER ligand-dependenttyrosine phosphorylation activity using an anti-phosphotyrosinemonoclonal (which is optionally conjugated with a detectable label). Thekinase receptor activation assay described in U.S. Pat. No. 5,766,863 isalso available for determining HER receptor activation and blocking ofthat activity by an antibody.

In one embodiment, one may screen for an antibody which inhibits HRGstimulation of p180 tyrosine phosphorylation in MCF7 cells essentiallyas described in WO01/00245. For example, the MCF7 cells may be plated in24-well plates and monoclonal antibodies to HER2 may be added to eachwell and incubated for 30 minutes at room temperature; thenrHRGβ1₁₇₇₋₂₄₄ may be added to each well to a final concentration of 0.2nM, and the incubation may be continued for 8 minutes. Media may beaspirated from each well, and reactions may be stopped by the additionof 100 μl of SDS sample buffer (5% SDS, 25 mM DTT, and 25 mM Tris-HCl,pH 6.8). Each sample (25 μA) may be electrophoresed on a 4-12% gradientgel (Novex) and then electrophoretically transferred to polyvinylidenedifluoride membrane. Antiphosphotyrosine (at 1 μg/ml) immunoblots may bedeveloped, and the intensity of the predominant reactive band atM_(r)˜180,000 may be quantified by reflectance densitometry. Theantibody selected will preferably significantly inhibit HRG stimulationof p180 tyrosine phosphorylation to about 0-35% of control in thisassay. A dose-response curve for inhibition of HRG stimulation of p180tyrosine phosphorylation as determined by reflectance densitometry maybe prepared and an IC₅₀ for the antibody of interest may be calculated.In one embodiment, the antibody which blocks ligand activation of a HERreceptor will have an IC₅₀ for inhibiting HRG stimulation of p180tyrosine phosphorylation in this assay of about 50 nM or less, morepreferably 10 nM or less. Where the antibody is an antibody fragmentsuch as a Fab fragment, the IC₅₀ for inhibiting HRG stimulation of p180tyrosine phosphorylation in this assay may, for example, be about 100 nMor less, more preferably 50 nM or less.

One may also assess the growth inhibitory effects of the antibody onMDA-MB-175 cells, e.g, essentially as described in Schaefer et al.Oncogene 15:1385-1394 (1997). According to this assay, MDA-MB-175 cellsmay treated with a HER2 monoclonal antibody (10 μg/mL) for 4 days andstained with crystal violet. Incubation with a HER2 antibody may show agrowth inhibitory effect on this cell line similar to that displayed bymonoclonal antibody 2C4. In a further embodiment, exogenous HRG will notsignificantly reverse this inhibition. Preferably, the antibody will beable to inhibit cell proliferation of MDA-MB-175 cells to a greaterextent than monoclonal antibody 4D5 (and optionally to a greater extentthan monoclonal antibody 7F3), both in the presence and absence ofexogenous HRG.

In one embodiment, the HER2 antibody of interest may block heregulindependent association of HER2 with HER3 in both MCF7 and SK-BR-3 cellsas determined in a co-immunoprecipitation experiment such as thatdescribed in WO01/00245 substantially more effectively than monoclonalantibody 4D5, and preferably substantially more effectively thanmonoclonal antibody 7F3.

To identify growth inhibitory HER2 antibodies, one may screen forantibodies which inhibit the growth of cancer cells which overexpressHER2. In one embodiment, the growth inhibitory antibody of choice isable to inhibit growth of SK-BR-3 cells in cell culture by about 20-100%and preferably by about 50-100% at an antibody concentration of about0.5 to 30 μg/ml. To identify such antibodies, the SK-BR-3 assaydescribed in U.S. Pat. No. 5,677,171 can be performed. According to thisassay, SK-BR-3 cells are grown in a 1:1 mixture of F12 and DMEM mediumsupplemented with 10% fetal bovine serum, glutamine and penicillinstreptomycin. The SK-BR-3 cells are plated at 20,000 cells in a 35 mmcell culture dish (2 mls/35 mm dish). 0.5 to 30 μg/ml of the HER2antibody is added per dish. After six days, the number of cells,compared to untreated cells are counted using an electronic COULTER™cell counter. Those antibodies which inhibit growth of the SK-BR-3 cellsby about 20-100% or about 50-100% may be selected as growth inhibitoryantibodies. See U.S. Pat. No. 5,677,171 for assays for screening forgrowth inhibitory antibodies, such as 4D5 and 3E8.

In order to select for antibodies which induce apoptosis, an annexinbinding assay using BT474 cells is available. The BT474 cells arecultured and seeded in dishes as discussed in the preceding paragraph.The medium is then removed and replaced with fresh medium alone ormedium containing 10 μg/ml of the monoclonal antibody. Following a threeday incubation period, monolayers are washed with PBS and detached bytrypsinization. Cells are then centrifuged, resuspended in Ca²⁺ bindingbuffer and aliquoted into tubes as discussed above for the cell deathassay. Tubes then receive labeled annexin (e.g. annexin V-FTIC) (1μg/ml). Samples may be analyzed using a FACSCAN™ flow cytometer andFACSCONVERT™ CellQuest software (Becton Dickinson). Those antibodieswhich induce statistically significant levels of annexin bindingrelative to control are selected as apoptosis-inducing antibodies. Inaddition to the annexin binding assay, a DNA staining assay using BT474cells is available. In order to perform this assay, BT474 cells whichhave been treated with the antibody of interest as described in thepreceding two paragraphs are incubated with 9 μg/ml HOECHST 33342™ for 2hr at 37° C., then analyzed on an EPICS ELITE™ flow cytometer (CoulterCorporation) using MODFIT LT™ software (Verity Software House).Antibodies which induce a change in the percentage of apoptotic cellswhich is 2 fold or greater (and preferably 3 fold or greater) thanuntreated cells (up to 100% apoptotic cells) may be selected aspro-apoptotic antibodies using this assay. See WO98/17797 for assays forscreening for antibodies which induce apoptosis, such as 7C2 and 7F3.

To screen for antibodies which bind to an epitope on HER2 bound by anantibody of interest, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed to assesswhether the antibody cross-blocks binding of an antibody, such as 2C4 orpertuzumab, to HER2. Alternatively, or additionally, epitope mapping canbe performed by methods known in the art and/or one can study theantibody-HER2 structure (Franklin et al. Cancer Cell 5:317-328 (2004))to see what domain(s) of HER2 is/are bound by the antibody.

(ix) Pertuzumab Compositions

In one embodiment of a HER2 antibody composition, the compositioncomprises a mixture of a main species pertuzumab antibody and one ormore variants thereof. The preferred embodiment herein of a pertuzumabmain species antibody is one comprising the variable light and variableheavy amino acid sequences in SEQ ID Nos. 3 and 4, and most preferablycomprising a light chain amino acid sequence selected from SEQ ID No. 13and 17, and a heavy chain amino acid sequence selected from SEQ ID No.14 and 18 (including deamidated and/or oxidized variants of thosesequences). In one embodiment, the composition comprises a mixture ofthe main species pertuzumab antibody and an amino acid sequence variantthereof comprising an amino-terminal leader extension. Preferably, theamino-terminal leader extension is on a light chain of the antibodyvariant (e.g. on one or two light chains of the antibody variant). Themain species HER2 antibody or the antibody variant may be an full lengthantibody or antibody fragment (e.g. Fab of F(ab′)2 fragments), butpreferably both are full length antibodies. The antibody variant hereinmay comprise an amino-terminal leader extension on any one or more ofthe heavy or light chains thereof. Preferably, the amino-terminal leaderextension is on one or two light chains of the antibody. Theamino-terminal leader extension preferably comprises or consists ofVHS-. Presence of the amino-terminal leader extension in the compositioncan be detected by various analytical techniques including, but notlimited to, N-terminal sequence analysis, assay for charge heterogeneity(for instance, cation exchange chromatography or capillary zoneelectrophoresis), mass spectrometry, etc. The amount of the antibodyvariant in the composition generally ranges from an amount thatconstitutes the detection limit of any assay (preferably N-terminalsequence analysis) used to detect the variant to an amount less than theamount of the main species antibody. Generally, about 20% or less (e.g.from about 1% to about 15%, for instance from 5% to about 15%) of theantibody molecules in the composition comprise an amino-terminal leaderextension. Such percentage amounts are preferably determined usingquantitative N-terminal sequence analysis or cation exchange analysis(preferably using a high-resolution, weak cation-exchange column, suchas a PROPAC WCX-10™ cation exchange column). Aside from theamino-terminal leader extension variant, further amino acid sequencealterations of the main species antibody and/or variant arecontemplated, including but not limited to an antibody comprising aC-terminal lysine residue on one or both heavy chains thereof, adeamidated antibody variant, etc.

Moreover, the main species antibody or variant may further compriseglycosylation variations, non-limiting examples of which includeantibody comprising a G1 or G2 oligosaccharide structure attached to theFc region thereof, antibody comprising a carbohydrate moiety attached toa light chain thereof (e.g. one or two carbohydrate moieties, such asglucose or galactose, attached to one or two light chains of theantibody, for instance attached to one or more lysine residues),antibody comprising one or two non-glycosylated heavy chains, orantibody comprising a sialidated oligosaccharide attached to one or twoheavy chains thereof etc.

The composition may be recovered from a genetically engineered cellline, e.g. a Chinese Hamster Ovary (CHO) cell line expressing the HER2antibody, or may be prepared by peptide synthesis.

(x) Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin(e.g. a small molecule toxin or an enzymatically active toxin ofbacterial, fungal, plant or animal origin, including fragments and/orvariants thereof), or a radioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Conjugates of an antibodyand one or more small molecule toxins, such as a calicheamicin, amaytansine (U.S. Pat. No. 5,208,020), a trichothene, and CC1065 are alsocontemplated herein.

In one preferred embodiment of the invention, the antibody is conjugatedto one or more maytansine molecules (e.g. about 1 to about 10 maytansinemolecules per antibody molecule). Maytansine may, for example, beconverted to May-SS-Me which may be reduced to May-SH3 and reacted withmodified antibody (Chari et al. Cancer Research 52: 127-131 (1992)) togenerate a maytansinoid-antibody immunoconjugate.

Another immunoconjugate of interest comprises an antibody conjugated toone or more calicheamicin molecules. The calicheamicin family ofantibiotics are capable of producing double-stranded DNA breaks atsub-picomolar concentrations. Structural analogues of calicheamicinwhich may be used include, but are not limited to, γ₁ ^(I), α₂ ^(I), α₃^(I), N-acetyl-γ₁ ^(I), PSAG and θ^(I) ₁ (Hinman et al. Cancer Research53: 3336-3342 (1993) and Lode et al. Cancer Research 58: 2925-2928(1998)). See, also, U.S. Pat. Nos. 5,714,586; 5,712,374; 5,264,586; and5,773,001 expressly incorporated herein by reference.

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g. aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

A variety of radioactive isotopes are available for the production ofradioconjugated HER2 antibodies. Examples include At²¹¹, I¹³¹, I¹²⁵,Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes of Lu.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al. Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, dimethyl linker or disulfide-containinglinker (Chari et al. Cancer Research 52: 127-131 (1992)) may be used.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g. by recombinant techniques or peptide synthesis.

Other immunoconjugates are contemplated herein. For example, theantibody may be linked to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, orcopolymers of polyethylene glycol and polypropylene glycol. The antibodyalso may be entrapped in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization (for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Oslo, A., Ed., (1980).

The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; andWO97/38731 published Oct. 23, 1997. Liposomes with enhanced circulationtime are disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al. J. Biol. Chem.257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al. J. National Cancer Inst. 81(19)1484 (1989).

III. Selecting Patients for Therapy

The patient herein is optionally subjected to a diagnostic test prior totherapy. For example, the diagnostic test may evaluate HER (e.g. HER2 orEGFR) expression (including overexpression), amplification, and/oractivation (including phosphorylation or dimerization).

Generally, if a diagnostic test is performed, a sample may be obtainedfrom a patient in need of therapy. Where the subject has cancer, thesample is generally a tumor sample. In the preferred embodiment, thetumor sample is from an ovarian cancer, peritoneal cancer, fallopiantube cancer, metastatic breast cancer (MBC), non-small cell lung cancer(NSCLC), prostate cancer, or colorectal cancer tumor sample.

It is noted however, that various other non-malignant therapeuticindications for HER antibodies are described herein. Where the patientis to be treated for those non-malignant indications, a suitable samplecan be obtained from the patient and subjected to a diagnostic assay asdescribed herein.

The biological sample herein may be a fixed sample, e.g. a formalinfixed, paraffin-embedded (FFPE) sample, or a frozen sample.

According to one embodiment of the invention herein, the patientselected for therapy has a tumor displaying HER (and preferably HER2)activation. In one embodiment, the extent of HER (or HER2) activation incancer cells significantly exceeds the level of activation of thatreceptor in non-cancerous cells of the same tissue type. Such excessiveactivation may result from overexpression of the HER receptor and/orgreater than normal levels of a HER ligand available for activating theHER receptor in the cancer cells. Such excessive activation may causeand/or be caused by the malignant state of a cancer cell. In someembodiments, the cancer will be subjected to a diagnostic or prognosticassay to determine whether amplification and/or overexpression of a HERreceptor is occurring which results in such excessive activation of theHER receptor. Alternatively, or additionally, the cancer may besubjected to a diagnostic or prognostic assay to determine whetheramplification and/or overexpression a HER ligand is occurring in thecancer which attributes to excessive activation of the receptor. In asubset of such cancers, excessive activation of the receptor may resultfrom an autocrine stimulatory pathway. Various assays for determiningHER activation will be described in more detail below.

(i) HER Dimers

Tumors samples can be assessed for the presence of HER dimers, asindicating HER or HER2 activation. Any method known in the art may beused to detect HER2 dimers, such as EGFR-HER2, HER2-HER3, in tumors.Several preferred methods are described below. These methods detectnoncovalent protein-protein interactions or otherwise indicate proximitybetween proteins of interest.

Immunoaffinity-based methods, such as immunoprecipitation or ELISA, maybe used to detect HER dimers. In one embodiment, HER2 antibodies areused to immunoprecipitate complexes comprising HER2 from tumor cells,and the resulting immunoprecipitant is then probed for the presence ofEGFR or HER3 by immunoblotting. In another embodiment, EGFR or HER3antibodies may be used for the immunoprecipitation step and theimmunoprecipitant then probed with HER2 antibodies. In a furtherembodiment, HER ligands specific to EGFR, HER3, EGFR/HER2 complexes orHER2/HER3 complexes may be used to precipitate complexes, which are thenprobed for the presence of HER2. For example, ligands may be conjugatedto avidin and complexes purified on a biotin column.

In other embodiments, such as ELISA or antibody “sandwich”-type assays,antibodies to HER2 are immobilized on a solid support, contacted withtumor cells or tumor cell lysate, washed, and then exposed to antibodyagainst EGFR or HER3. Binding of the latter antibody, which may bedetected directly or by a secondary antibody conjugated to a detectablelabel, indicates the presence of heterodimers. In certain embodiments,EGFR or HER3 antibody is immobilized, and HER2 antibody is used for thedetection step. In other embodiments HER ligands may be used in placeof, or in combination with HER antibodies.

Chemical or UV cross-linking may also be used to covalently join dimerson the surface of living cells. Hunter et al., Biochem. J., 320:847-53.Examples of chemical cross-linkers include dithiobis(succinimidyl)propionate (DSP) and 3,3′dithiobis(sulphosuccinimidyl)propionate(DTSSP). In one embodiment, cell extracts from chemically cross-linkedtumor cells are analyzed by SDS-PAGE and immunoblotted with antibodiesto EGFR and/or HER3. A supershifted band of the appropriate molecularweight most likely represents EGFR-HER2 or HER2-HER3 dimers, as HER2 isthe preferred dimerization partner for EGFR and HER3. This result may beconfirmed by subsequent immunoblotting with HER2 antibodies.

Fluorescence resonance energy transfer (FRET) may also be used to detectEGFR-HER2 or HER2-HER3 dimers. FRET detects protein conformationalchanges and protein-protein interactions in vivo and in vitro based onthe transfer of energy from a donor fluorophore to an acceptorfluorophore. Selvin, Nat. Struct. Biol. 7:730-34 (2000). Energy transfertakes place only if the donor fluorophore is in sufficient proximity tothe acceptor fluorophore. In a typical FRET experiment, two proteins ortwo sites on a single protein are labeled with different fluorescentprobes. One of the probes, the donor probe, is excited to a higherenergy state by incident light of a specified wavelength. The donorprobe then transmits its energy to the second probe, the acceptor probe,resulting in a reduction in the donor's fluorescence intensity and anincrease in the acceptor's fluorescence emission. To measure the extentof energy transfer, the donor's intensity in a sample labeled with donorand acceptor probes is compared with its intensity in a sample labeledwith donor probe only. Optionally, acceptor intensity is compared indonor/acceptor and acceptor only samples. Suitable probes are known inthe art and include, for example, membrane permeant dyes, such asfluorescein and rhodamine, organic dyes, such as the cyanine dyes, andlanthanide atoms. Selvin, supra. Methods and instrumentation fordetecting and measuring energy transfer are also known in the art.Selvin, supra.

FRET-based techniques suitable for detecting and measuringprotein-protein interactions in individual cells are also known in theart. For example, donor photobleaching fluorescence resonance energytransfer (pbFRET) microscopy and fluorescence lifetime imagingmicroscopy (FLIM) may be used to detect the dimerization of cell surfacereceptors. Selvin, supra; Gadella & Jovin, J. Cell Biol., 129:1543-58(1995). In one embodiment, pbFRET is used on cells either “insuspension” or “in situ” to detect and measure the formation ofEGFR-HER2 or HER2-HER3 dimers, as described in Nagy et al., Cytometry,32:120-131 (1998). These techniques measure the reduction in a donor'sfluorescence lifetime due to energy transfer. In a particularembodiment, a flow cytometric Foerster-type FRET technique (FCET) may beused to investigate EGFR-HER2 and HER2-HER3 dimerization, as describedin Nagy et al., supra, and Brockhoff et al., Cytometry, 44:338-48(2001).

FRET is preferably used in conjunction with standard immunohistochemicallabeling techniques. Kenworthy, Methods, 24:289-96 (2001). For example,antibodies conjugated to suitable fluorescent dyes can be used as probesfor labeling two different proteins. If the proteins are withinproximity of one another, the fluorescent dyes act as donors andacceptors for FRET. Energy transfer is detected by standard means.Energy transfer may be detected by flow cytometric means or by digitalmicroscopy systems, such as confocal microscopy or wide-fieldfluorescence microscopy coupled to a charge-coupled device (CCD) camera.

In one embodiment of the present invention, HER2 antibodies and eitherEGFR or HER3 antibodies are directly labeled with two differentfluorophores, for example as described in Nagy et al, supra. Tumor cellsor tumor cell lysates are contacted with the differentially labeledantibodies, which act as donors and acceptors for FRET in the presenceof EGFR-HER2 or HER2-HER3 dimers. Alternatively, unlabeled antibodiesagainst HER2 and either EGFR or HER3 are used along with differentiallylabeled secondary antibodies that serve as donors and acceptors. See,for example, Brockhoff et al., supra. Energy transfer is detected andthe presence of dimers is determined if the labels are found to be inclose proximity.

In other embodiments HER receptor ligands that are specific for HER2 andeither HER1 or HER3 are fluorescently labeled and used for FRET studies.

In still other embodiments of the present invention, the presence ofdimers on the surface of tumor cells is demonstrated by co-localizationof HER2 with either EGFR or HER3 using standard direct or indirectimmunofluorescence techniques and confocal laser scanning microscopy.Alternatively, laser scanning imaging (LSI) is used to detect antibodybinding and co-localization of HER2 with either EGFR or HER3 in ahigh-throughput format, such as a microwell plate, as described in Zucket al, Proc. Natl. Acad. Sci. USA, 96:11122-27 (1999).

In further embodiments, the presence of EGFR-HER2 and/or HER2-HER3dimers is determined by identifying enzymatic activity that is dependentupon the proximity of the dimer components. A HER2 antibody isconjugated with one enzyme and an EGFR or HER3 antibody is conjugatedwith a second enzyme. A first substrate for the first enzyme is addedand the reaction produces a second substrate for the second enzyme. Thisleads to a reaction with another molecule to produce a detectablecompound, such as a dye. The presence of another chemical breaks downthe second substrate, so that reaction with the second enzyme isprevented unless the first and second enzymes, and thus the twoantibodies, are in close proximity. In a particular embodiment tumorcells or cell lysates are contacted with a HER2 antibody that isconjugated with glucose oxidase and a HER3 or HER1 antibody that isconjugated with horse radish peroxidase. Glucose is added to thereaction, along with a dye precursor, such as DAB, and catalase. Thepresence of dimers is determined by the development of color uponstaining for DAB.

Dimers may also be detected using methods based on the eTag™ assaysystem (Aclara Bio Sciences, Mountain View, Calif.), as described, forexample, in U.S. Patent Application 2001/0049105, published Dec. 6,2001, both of which are expressly incorporated by reference in theirentirety. An eTag™, or “electrophoretic tag,” comprises a detectablereporter moiety, such as a fluorescent group. It may also comprise a“mobility modifier,” which consists essentially of a moiety having aunique electrophoretic mobility. These moieties allow for separation anddetection of the eTag™ from a complex mixture under definedelectrophoretic conditions, such as capillary electrophoresis (CE). Theportion of the eTag™ containing the reporter moiety and, optionally, themobility modifier is linked to a first target binding moiety by acleavable linking group to produce a first binding compound. The firsttarget binding moiety specifically recognizes a particular first target,such as a nucleic acid or protein. The first target binding moiety isnot limited in any way, and may be for example, a polynucleotide or apolypeptide. Preferably, the first target binding moiety is an antibodyor antibody fragment. Alternatively, the first target binding moiety maybe a HER receptor ligand or binding-competent fragment thereof.

The linking group preferably comprises a cleavable moiety, such as anenzyme substrate, or any chemical bond that may be cleaved under definedconditions. When the first target binding moiety binds to its target,the cleaving agent is introduced and/or activated, and the linking groupis cleaved, thus releasing the portion of the eTag™ containing thereporter moiety and mobility modifier. Thus, the presence of a “free”eTag™ indicates the binding of the target binding moiety to its target.

Preferably, a second binding compound comprises the cleaving agent and asecond target binding moiety that specifically recognizes a secondtarget. The second target binding moiety is also not limited in any wayand may be, for example, an antibody or antibody fragment or a HERreceptor ligand or binding competent ligand fragment. The cleaving agentis such that it will only cleave the linking group in the first bindingcompound if the first binding compound and the second binding compoundare in close proximity.

In an embodiment of the present invention, a first binding compoundcomprises an eTag™ in which an antibody to HER2 serves as the firsttarget binding moiety. A second binding compound comprises an antibodyto EGFR or HER3 joined to a cleaving agent capable of cleaving thelinking group of the eTag™. Preferably the cleaving agent must beactivated in order to be able to cleave the linking group. Tumor cellsor tumor cell lysates are contacted with the eTag™, which binds to HER2,and with the modified EGFR or HER3 antibody, which binds to EGFR or HER3on the cell surface. Unbound binding compound is preferable removed, andthe cleaving agent is activated, if necessary. If EGFR-HER2 or HER2-HER3dimers are present, the cleaving agent will cleave the linking group andrelease the eTag™ due to the proximity of the cleaving agent to thelinking group. Free eTag™ may then be detected by any method known inthe art, such as capillary electrophoresis.

In one embodiment, the cleaving agent is an activatable chemical speciesthat acts on the linking group. For example, the cleaving agent may beactivated by exposing the sample to light.

In another embodiment, the eTag™ is constructed using an antibody toEGFR or HER3 as the first target binding moiety, and the second bindingcompound is constructed from an antibody to HER2.

In yet another embodiment, the HER dimer is detected using an antibodyor other reagent which specifically or preferentially binds to the dimeras compared to binding thereof to either HER receptor in the dimer.

(ii) HER2 Phosphorylation

Immunoprecipitation with EGFR, HER2, or HER3 antibody as discussed inthe previous section may optionally be followed by a functional assayfor dimers, as an alternative or supplement to immunoblotting. In oneembodiment, immunoprecipitation with HER3 antibody is followed by anassay for receptor tyrosine kinase activity in the immunoprecipitant.Because HER3 does not have intrinsic tyrosine kinase activity, thepresence of tyrosine kinase activity in the immunoprecipitant indicatesthat HER3 is most likely associated with HER2. Graus-Porta et al., EMBOJ., 16:1647-55 (1997); Klapper et al., Proc. Natl. Acad. Sci. USA,96:4995-5000 (1999). This result may be confirmed by immunoblotting withHER2 antibodies. In another embodiment, immunoprecipitation with HER2antibody is followed by an assay for EGFR receptor tyrosine kinaseactivity. In this assay, the immunoprecipitant is contacted withradioactive ATP and a peptide substrate that mimics the in vivo site oftransphosphorylation of HER2 by EGFR. Phosphorylation of the peptideindicates co-immunoprecipitation and thus dimerization of EGFR withHER2. Receptor tyrosine kinase activity assays are well known in the artand include assays that detect phosphorylation of target substrates, forexample, by phosphotyrosine antibody, and activation of cognate signaltransduction pathways, such as the MAPK pathway.

Phosphorylation of HER receptor may be assessed by immunoprecipitationof one or more HER receptors, such as HER2 receptor, and Western blotanalysis. For example, positivity is determined by the presence of aphospho-HER2 band on the gel, using an anti-phosphotyrosine antibody todetect phosphorylated tyrosine residue(s) in the immunoprecipitated HERreceptor(s). Anti-phosphotyrosine antibodies are commercially availablefrom PanVera (Madison, Wis.), a subsidiary of Invitrogen, ChemiconInternational Inc. (Temecula, Calif.), or Upstate Biotechnology (LakePlacid, N.Y.). Negativity is determined by the absence of the band.

In another embodiment, phosphorylation of HER2 (HER2) receptor isassessed by immunohistochemistry using a phospho-specific HER2 antibody(clone PN2A; Thor et al., J. Clin. Oncol, 18(18):3230-3239 (2000)).

Other methods for detecting phosphorylation of HER receptor(s) include,but are not limited to, KIRA ELISA (U.S. Pat. Nos. 5,766,863; 5,891,650;5,914,237; 6,025,145; and 6,287,784), mass spectrometry (comparing sizeof phosphorylated and non-phosphorylated HER2), and e-tag proximityassay with both a HER (e.g. HER2) antibody and phospho-specific orphospho-tyrosine specific antibody (e.g., using the eTag™ assay kitavailable from Aclara BioSciences (Mountain View, Calif.). Details ofthe eTag assay are described hereinabove.

One may also use phospho-specific antibodies in cellular array to detectphosphorylation status in a cellular sample of signal transductionprotein (US2003/0190689).

(iii) Gene Expression Profile

In one embodiment, a gene expression analyses can serve as a surrogatefor measuring HER phosphorylation or activation directly. This isparticularly useful where the sample is a fixed sample (e.g.parrafin-embedded, formalin fixed tumor sample) where HERphosphorylation may be difficult to reliably quantify. According to thismethod, expression of two or more HER receptors and one or more HERligand in a sample is evaluated, wherein expression of the two or moreHER receptors and one or more HER ligand indicates positive HERphosphorylation or activation in the sample. In one embodiment of thismethod, expression of betacellulin and/or amphiregulin in the sample canbe measured, wherein betacellulin and/or amphiregulin expressionindicates positive HER phosphorylation or activation in the sample.

According to this method, a sample from the patient is tested forexpression of two or more HER receptors (preferably selected from EGFR,HER2, and HER3) and one or more HER ligands (preferably selected frombetacellulin, amphiregulin, epiregulin, and TGF-α, most preferablybetacellulin or amphiregulin). For example, the two or more HERreceptors may be EGFR and HER2, or HER2 and HER3. Preferably, expressionof HER2 and EGFR or HER3, as well as betacellulin or amphiregulin isdetermined. The sample may be tested for expression of betacellulin oramphiregulin alone, or in combination with testing for expression of twoor more HER receptors. Positive expression of the identified gene(s)indicates the patient is a candidate for therapy with a HER antibody,such as pertuzumab. Moreover, positive expression of the gene(s)indicates the patient is more likely to respond favorably to therapywith the HER antibody than a patient who does not have such positiveexpression.

Various methods for determining expression of mRNA or protein include,but are not limited to, gene expression profiling, polymerase chainreaction (PCR) including quantitative real time PCR (qRT-PCR),microarray analysis, serial analysis of gene expression (SAGE),MassARRAY, Gene Expression Analysis by Massively Parallel SignatureSequencing (MPSS), proteomics, immunohistochemistry (1HC), etc.Preferably mRNA is quantified. Such mRNA analysis is preferablyperformed using the technique of polymerase chain reaction (PCR), or bymicroarray analysis. Where PCR is employed, a preferred form of PCR isquantitative real time PCR (qRT-PCR). In one embodiment, expression ofone or more of the above noted genes is deemed positive expression if itis at the median or above, e.g. compared to other samples of the sametumor-type. The median expression level can be determined essentiallycontemporaneously with measuring gene expression, or may have beendetermined previously.

Various exemplary methods for determining gene expression The steps of arepresentative protocol for profiling gene expression using fixed,paraffin-embedded tissues as the RNA source, including mRNA isolation,purification, primer extension and amplification are given in variouspublished journal articles (for example: Godfrey et al. J. Molec.Diagnostics 2: 84-91 (2000); Specht et al., Am. J. Pathol. 158: 419-29(2001)). Briefly, a representative process starts with cutting about 10microgram thick sections of paraffin-embedded tumor tissue samples. TheRNA is then extracted, and protein and DNA are removed. After analysisof the RNA concentration, RNA repair and/or amplification steps may beincluded, if necessary, and RNA is reverse transcribed using genespecific promoters followed by PCR. Finally, the data are analyzed toidentify the best treatment option(s) available to the patient on thebasis of the characteristic gene expression pattern identified in thetumor sample examined.

(iv) HER Expression and Amplification

To determine HER expression or amplification in the cancer, variousdiagnostic/prognostic assays are available. In one embodiment, HERoverexpression may be analyzed by IHC, e.g. using the HERCEPTEST®(Dako).Parrafin embedded tissue sections from a tumor biopsy may be subjectedto the IHC assay and accorded a HER2 protein staining intensity criteriaas follows:

Score 0 no staining is observed or membrane staining is observed in lessthan 10% of tumor cells.Score 1+ a faint/barely perceptible membrane staining is detected inmore than 10% of the tumor cells. The cells are only stained in part oftheir membrane.Score 2+ a weak to moderate complete membrane staining is observed inmore than 10% of the tumor cells.Score 3+ a moderate to strong complete membrane staining is observed inmore than 10% of the tumor cells.

Those tumors with 0 or 1+ scores for HER2 overexpression assessment maybe characterized as not overexpressing HER2, whereas those tumors with2+ or 3+ scores may be characterized as overexpressing HER2.

Tumors overexpressing HER2 may be rated by immunohistochemical scorescorresponding to the number of copies of HER2 molecules expressed percell, and can been determined biochemically:

0=0-10,000 copies/cell,1+=at least about 200,000 copies/cell,2+=at least about 500,000 copies/cell,3+=at least about 2,000,000 copies/cell.

Overexpression of HER2 at the 3+ level, which leads toligand-independent activation of the tyrosine kinase (Hudziak et al.,Proc. Natl. Acad. Sci. USA, 84:7159-7163 (1987)), occurs inapproximately 30% of breast cancers, and in these patients, relapse-freesurvival and overall survival are diminished (Slamon et al., Science,244:707-712 (1989); Slamon et al., Science, 235:177-182 (1987)).

Alternatively, or additionally, FISH assays such as the INFORM™ (sold byVentana, Ariz.) or PATHVISION™ (Vysis, Ill.) may be carried out onformalin-fixed, paraffin-embedded tumor tissue to determine the extent(if any) of HER2 amplification in the tumor.

In one embodiment, the cancer will be one which expresses (and mayoverexpress) EGFR, such expression may be evaluated as for the methodsfor evaluating HER2 expression as noted above.

HER receptor or HER ligand overexpression or amplification may also beevaluated using an in vivo diagnostic assay, e.g. by administering amolecule (such as an antibody) which binds the molecule to be detectedand is tagged with a detectable label (e.g. a radioactive isotope) andexternally scanning the patient for localization of the label.

IV. Pharmaceutical Formulations

Therapeutic formulations of the antibodies used in accordance with thepresent invention are prepared for storage by mixing an antibody havingthe desired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Lyophilized antibody formulations aredescribed in WO 97/04801, expressly incorporated herein by reference.

The preferred pertuzumab formulation for therapeutic use comprises 30mg/mL pertuzumab in 20 mM histidine acetate, 120 mM sucrose, 0.02%polysorbate 20, at pH 6.0. An alternate pertuzumab formulation comprises25 mg/mL pertuzumab, 10 mM histidine-HCl buffer, 240 mM sucrose, 0.02%polysorbate 20, pH 6.0.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Various drugs which can be combined with the HER antibody are describedin the method of treatment section below. Such molecules are suitablypresent in combination in amounts that are effective for the purposeintended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

V. Treatment and Dosing of HER Antibodies

Examples of various cancers that can be treated with a fixed dose of aHER antibody are listed in the definition section above. Preferredcancer indications include ovarian cancer; peritoneal cancer; fallopiantube cancer; breast cancer, including metastatic breast cancer (MBC);lung cancer, including non-small cell lung cancer (NSCLC); prostatecancer; colorectal cancer; and/or cancer which displays HER expression,amplification and/or activation. In one embodiment, the cancer which istreated is chemotherapy-resistant cancer or platinum-resistant cancer.Administration of fixed dose(s) of the antibody will result in animprovement in the signs or symptoms of cancer.

Aside from cancer, fixed dose(s) of the HER antibodies as disclosedherein may be used to treat various non-malignant diseases or disorders.Such non-malignant diseases or disorders include autoimmune disease(e.g. psoriasis; see definition above); endometriosis; scleroderma;restenosis; polyps such as colon polyps, nasal polyps orgastrointestinal polyps; fibroadenoma; respiratory disease (seedefinition above); cholecystitis; neurofibromatosis; polycystic kidneydisease; inflammatory diseases; skin disorders including psoriasis anddermatitis; vascular disease (see definition above); conditionsinvolving abnormal proliferation of vascular epithelial cells;gastrointestinal ulcers; Menetrier's disease, secreting adenomas orprotein loss syndrome; renal disorders; angiogenic disorders; oculardisease such as age related macular degeneration, presumed ocularhistoplasmosis syndrome, retinal neovascularization from proliferativediabetic retinopathy, retinal vascularization, diabetic retinopathy, orage related macular degeneration; bone associated pathologies such asosteoarthritis, rickets and osteoporosis; damage following a cerebralischemic event; fibrotic or edemia diseases such as hepatic cirrhosis,lung fibrosis, carcoidosis, throiditis, hyperviscosity syndromesystemic, Osler Weber-Rendu disease, chronic occlusive pulmonarydisease, or edema following burns, trauma, radiation, stroke, hypoxia orischemia; hypersensitivity reaction of the skin; diabetic retinopathyand diabetic nephropathy; Guillain-Barre syndrome; graft versus hostdisease or transplant rejection; Paget's disease; bone or jointinflammation; photoaging (e.g. caused by UV radiation of human skin);benign prostatic hypertrophy; certain microbial infections includingmicrobial pathogens selected from adenovirus, hantaviruses, Borreliaburgdorferi, Yersinia spp. and Bordetella pertussis; thrombus caused byplatelet aggregation; reproductive conditions such as endometriosis,ovarian hyperstimulation syndrome, preeclampsia, dysfunctional uterinebleeding, or menometrorrhagia; synovitis; atheroma; acute and chronicnephropathies (including proliferative glomerulonephritis anddiabetes-induced renal disease); eczema; hypertrophic scar formation;endotoxic shock and fungal infection; familial adenomatosis polyposis;neurodedenerative diseases (e.g. Alzheimer's disease, AIDS-relateddementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitispigmentosa, spinal muscular atrophy and cerebellar degeneration);myelodysplastic syndromes; aplastic anemia; ischemic injury; fibrosis ofthe lung, kidney or liver; T-cell mediated hypersensitivity disease;infantile hypertrophic pyloric stenosis; urinary obstructive syndrome;psoriatic arthritis; and Hasimoto's thyroiditis. Preferred non-malignantindications for therapy herein include psoriasis, endometriosis,scleroderma, vascular disease (e.g. restenosis, artherosclerosis,coronary artery disease, or hypertension), colon polyps, fibroadenoma orrespiratory disease (e.g. asthma, chronic bronchitis, bronchieactasis orcystic fibrosis).

The HER antibody is administered to a human patient in accord with knownmethods, such as intravenous administration, e.g., as a bolus or bycontinuous infusion over a period of time, by intramuscular,intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes.Intravenous administration of the antibody is preferred.

For the prevention or treatment of disease, the fixed dose of HERantibody will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The fixed dose is suitablyadministered to the patient at one time or over a series of treatments.Preferably, the fixed dose is in the range from about 20 mg to about2000 mg of the HER antibody. For example, the fixed dose may beapproximately 420 mg, approximately 525 mg, approximately 840 mg, orapproximately 1050 mg of the HER antibody.

Where a series of fixed doses are administered, these may, for example,be administered approximately every week, approximately every 2 weeks,approximately every 3 weeks, or approximately every 4 weeks, butpreferably approximately every 3 weeks. The fixed doses may, forexample, continue to be administered until disease progression, adverseevent, or other time as determined by the physician. For example, fromabout two, three, or four, up to about 17 or more fixed doses may beadministered.

In one embodiment, one or more loading dose(s) of the antibody areadministered, followed by one or more maintenance dose(s) of theantibody. In another embodiment, a plurality of the same fixed dose areadministered to the patient.

According to one preferred embodiment of the invention, a fixed dose ofHER antibody (e.g. pertuzumab) of approximately 840 mg (loading dose) isadministered, followed by one or more doses of approximately 420 mg(maintenance dose(s)) of the antibody. The maintenance doses arepreferably administered about every 3 weeks, for a total of at least twodoses, up to 17 or more doses.

According to another preferred embodiment of the invention, one or morefixed dose(s) of approximately 1050 mg of the HER antibody (e.g.pertzumab) are administered, for example every 3 weeks. According tothis embodiment, one, two or more of the fixed doses are administered,e.g. for up to one year (17 cycles), and longer as desired.

In another embodiment, a fixed dose of approximately 1050 mg of HER2antibody (e.g. pertuzumab) is administered as a loading dose, followedby one or more maintenance dose(s) of approximately 525 mg of theantibody. About one, two or more maintenance doses may be administeredto the patient every 3 weeks according to this embodiment.

Thus, the invention provides a method of treating cancer in a humanpatient comprising administering at least one fixed dose of pertuzumabto the patient, wherein the fixed dose is approximately 420 mg,approximately 525 mg, approximately 840 mg, or approximately 1050 mg ofpertuzumab.

Where the disease is cancer, the patient is preferably treated with acombination of the HER antibody, and one or more chemotherapeuticagent(s). Preferably at least one of the chemotherapeutic agents is anantimetabolite chemotherapeutic agent such as gemcitabine. The combinedadministration includes coadministration or concurrent administration,using separate formulations or a single pharmaceutical formulation, andconsecutive administration in either order, wherein preferably there isa time period while both (or all) active agents simultaneously exerttheir biological activities. Thus, the antimetabolite chemotherapeuticagent may be administered prior to, or following, administration of theHER antibody. In this embodiment, the timing between at least oneadministration of the antimetabolite chemotherapeutic agent and at leastone administration of the HER antibody is preferably approximately 1month or less, and most preferably approximately 2 weeks or less.Alternatively, the antimetabolite chemotherapeutic agent and the HERantibody are administered concurrently to the patient, in a singleformulation or separate formulations. Treatment with the combination ofthe chemotherapeutic agent (e.g. antimetabolite chemotherapeutic agentsuch as gemcitabine) and the HER antibody (e.g. pertuzumab) may resultin a synergistic, or greater than additive, therapeutic benefit to thepatient.

An antimetabolite chemotherapeutic agent, if administered, is usuallyadministered at dosages known therefor, or optionally lowered due tocombined action of the drugs or negative side effects attributable toadministration of the antimetabolite chemotherapeutic agent. Preparationand dosing schedules for such chemotherapeutic agents may be usedaccording to manufacturers' instructions or as determined empirically bythe skilled practitioner. Where the antimetabolite chemotherapeuticagent is gemcitabine, preferably, it is administered at a dose betweenabout 600 mg/m² to 1250 mg/m² (for example approximately 1000 mg/m²),for instance, on days 1 and 8 of a 3-week cycle.

Aside from the HER antibody and antimetabolite chemotherapeutic agent,other therapeutic regimens may be combined therewith. For example, asecond (third, fourth, etc) chemotherapeutic agent(s) may beadministered, wherein the second chemotherapeutic agent is eitheranother, different antimetabolite chemotherapeutic agent, or achemotherapeutic agent that is not an antimetabolite. For example, thesecond chemotherapeutic agent may be a taxane (such as paclitaxel ordocetaxel), capecitabine, or platinum-based chemotherapeutic agent (suchas carboplatin, cisplatin, or oxaliplatin), anthracycline (such asdoxorubicin, including, liposomal doxorubicin), topotecan, pemetrexed,vinca alkaloid (such as vinorelbine), and TLK 286. “Cocktails” ofdifferent chemotherapeutic agents may be administered.

Other therapeutic agents that may be combined with the HER antibodyinclude any one or more of: a second, different HER antibody (forexample, a growth inhibitory HER2 antibody such as trastuzumab, or aHER2 antibody which induces apoptosis of a HER2-overexpressing cell,such as 7C2, 7F3 or humanized variants thereof); an antibody directedagainst a different tumor associated antigen, such as EGFR, HER3, HER4;anti-hormonal compound, e.g., an anti-estrogen compound such astamoxifen, or an aromatase inhibitor; a cardioprotectant (to prevent orreduce any myocardial dysfunction associated with the therapy); acytokine; an EGFR-targeted drug (such as TARCEVA®, IRESSA® orCetuximab); an anti-angiogenic agent (especially Bevacizumab sold byGenentech under the trademark AVASTIN™); a tyrosine kinase inhibitor; aCOX inhibitor inhibitor (for instance a COX-1 or COX-2 inhibitor);non-steroidal anti-inflammatory drug, Celecoxib (CELEBREX®); farnesyltransferase inhibitor (for example, Tipifarnib/ZARNESTRA® R115777available from Johnson and Johnson or Lonafarnib SCH66336 available fromSchering-Plough); antibody that binds oncofetal protein CA 125 such asOregovomab (MoAb B43.13); HER2 vaccine (such as HER2 AutoVac vaccinefrom Pharmexia, or APC8024 protein vaccine from Dendreon, or HER2peptide vaccine from GSK/Corixa); another HER targeting therapy (e.g.trastuzumab, cetuximab, gefitinib, erlotinib, CI1033, GW2016 etc); Rafand/or ras inhibitor (see, for example, WO 2003/86467); doxorubicin HClliposome injection (DOXIL®); topoisomerase I inhibitor such astopotecan; taxane; HER2 and EGFR dual tyrosine kinase inhibitor such aslapatinib/GW572016; TLK286 (TELCYTA®); EMD-7200; a medicament thattreats nausea such as a serotonin antagonist, steroid, orbenzodiazepine; a medicament that prevents or treats skin rash orstandard acne therapies, including topical or oral antibiotic; a bodytemperature-reducing medicament such as acetaminophen, diphenhydramine,or meperidine; hematopoietic growth factor, etc.

Suitable dosages for any of the above coadministered agents are thosepresently used and may be lowered due to the combined action (synergy)of the agent and HER antibody.

In addition to the above therapeutic regimes, the patient may besubjected to surgical removal of cancer cells and/or radiation therapy.

Preferably, the antibody administered is a naked antibody. However, theantibody administered may be conjugated with a cytotoxic agent.Preferably, the immunoconjugate and/or antigen to which it is boundis/are internalized by the cell, resulting in increased therapeuticefficacy of the immunoconjugate in killing the cancer cell to which itbinds. In a preferred embodiment, the cytotoxic agent targets orinterferes with nucleic acid in the cancer cell. Examples of suchcytotoxic agents include maytansinoids, calicheamicins, ribonucleasesand DNA endonucleases.

Aside from administration of the antibody protein to the patient, thepresent application contemplates administration of an antibody orprotein inhibitor by gene therapy. See, for example, WO96/07321published Mar. 14, 1996 concerning the use of gene therapy to generateintracellular antibodies.

There are two major approaches to getting the nucleic acid (optionallycontained in a vector) into the patient's cells; in vivo and ex vivo.For in vivo delivery the nucleic acid is injected directly into thepatient, usually at the site where the antibody is required. For ex vivotreatment, the patient's cells are removed, the nucleic acid isintroduced into these isolated cells and the modified cells areadministered to the patient either directly or, for example,encapsulated within porous membranes which are implanted into thepatient (see, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187). There are avariety of techniques available for introducing nucleic acids intoviable cells. The techniques vary depending upon whether the nucleicacid is transferred into cultured cells in vitro, or in vivo in thecells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. A commonly used vector for ex vivodelivery of the gene is a retrovirus.

The currently preferred in vivo nucleic acid transfer techniques includetransfection with viral vectors (such as adenovirus, Herpes simplex Ivirus, or adeno-associated virus) and lipid-based systems (useful lipidsfor lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, forexample). In some situations it is desirable to provide the nucleic acidsource with an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87:3410-3414 (1990). For review of the currently knowngene marking and gene therapy protocols see Anderson et al., Science256:808-813 (1992). See also WO 93/25673 and the references citedtherein.

VI. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of cancer or the otherdisorders described above is provided. The article of manufacturecomprises a vial with a fixed dose of the HER antibody contained thereinand, optionally, a package insert. The vial may be formed from a varietyof materials such as glass or plastic, and may be sealed by a stopperpierceable by a syringe. For example, the vial may be a formal vitrumtype I glass vial (e.g. 20 cc vial for a 420 mg fixed dose or 50 cc vialfor a 1050 mg fixed dose), with DAIKYO GREY™ fluoro-resin laminatedstopper, and 20 mm flip top aluminum cap. The article of manufacture mayfurther include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles, andsyringes, etc.

In the preferred embodiment, the article of manufacture comprises a vialcontaining a fixed dose of a HER antibody (e.g. pertuzumab), wherein thefixed dose is approximately 420 mg, approximately 525 mg, approximately840 mg, or approximately 1050 mg of the HER antibody.

The article of manufacture preferably further comprises a packageinsert. The package insert may provide instructions to administer thefixed dose to a cancer patient, including but not limited to a patientwith ovarian cancer, peritoneal cancer, fallopian tube cancer,metastatic breast cancer (MBC), non-small cell lung cancer (NSCLC),prostate cancer, colorectal cancer, and/or to administer the fixed doseto a cancer patient whose cancer displays HER expression, amplificationand/or phosphorylation.

In one embodiment, the article of manufacture comprises two vials,wherein a first vial contains a fixed dose of approximately 840 mg ofpertuzumab, and a second vial contains a fixed dose of approximately 420mg of pertuzumab.

In another embodiment, the article of manufacture of comprises twovials, wherein a first vial contains a fixed dose of approximately 1050mg of pertuzumab, and a second vial contains a fixed dose ofapproximately 525 mg of pertuzumab.

VII. Deposit of Materials

The following hybridoma cell lines have been deposited with the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, USA (ATCC):

Antibody Designation ATCC No. Deposit Date 7C2 ATCC HB-12215 Oct. 17,1996 7F3 ATCC HB-12216 Oct. 17, 1996 4D5 ATCC CRL 10463 May 24, 1990 2C4ATCC HB-12697 Apr. 8, 1999

Further details of the invention are illustrated by the followingnon-limiting Examples. The disclosures of all citations in thespecification are expressly incorporated herein by reference.

Example 1

The present example evaluates the population pharmacokinetic (PK) andpredictive covariates for the HER antibody pertuzumab, and examined thevariability of steady-state trough serum concentrations after fixed,body weight-based, or BSA-based dosing methods. Pertuzumab wasadministered by IV infusion (q3 week) either as a weight-based dose(0.5-15 mg/kg) or a fixed dose (420 mg or 1050 mg). Pertuzumab serumconcentration data from one phase Ia and two phase II trials (ovarianand breast), comprising 153 patients and 1458 concentration-time points,were pooled for this analysis using NONMEM™ with the first-orderconditional estimation with Interaction (FOCE interaction) method. Alinear 2-compartment model best described the data. Body weight, serumalbumin, and serum alkaline phosphatase were significant covariatesaffecting clearance (CL), and body-surface area (BSA) was a significantvariable affecting distribution volume at central compartment (Vc). Inthe final model, CL and Vc were 0.214 L/day and 2.74 L, respectively.Weight only explained 8.3% of inter-patient variability for CL.Evaluation of the final population PK model using a posterior predictivecheck showed good performance. Compared to fixed dosing, weight- andBSA-based dosing only reduced the population variability of steady-statetrough concentrations by 6.2% and 5.8%, respectively, in 1000 simulatedsubjects bootstrapped from the original data set using the final model.Simulations also showed that the percentages of subjects with predictedsteady-state trough concentrations below a target of 20 mcg/mL weresimilar following fixed, weight- or BSA-based dosing. It was concludedthat although humanized antibodies are typically dosed by weight, theanalyses in this example demonstrate the desirability of administeringthe HER antibody, pertuzumab, using a fixed dose to treat cancer.

Methods Studies and Patients

All three studies used in this analysis were approved by the appropriateethics committees of the participating centers. Written informed consentwas obtained from all patients.

Study 1 was a Phase Ia, open-label, multicenter, dose-escalation studyto evaluate the safety, tolerability, and pharmacokinetic profiles ofpertuzumab administered intravenously as a single agent to subjects withadvanced solid malignancies. These patients received a dose ofpertuzumab administered by the IV route every 3 weeks as a 90-minute IVinfusion on the 1^(st) cycle, then as 30-minute infusion in subsequentcycles. Doses were escalated (0.5, 2, 5, 10, and 15 mg/kg) in cohorts of3 or 6 subjects until the maximum tolerated dose (MTD) was defined orthe highest dose level was reached. During the first cycle of treatment,serum samples for determination of pertuzumab concentrations werecollected at serial time points: prior to the dose, at the end of the IVinfusion, at 1.5, 4, and 9 hours, and on days 2, 5, 8, and 15. Duringthe second treatment cycle, serum samples for determination ofpertuzumab concentrations were collected prior to the dose, 29 minutesfollowing the start of the IV infusion, and on day 8.

Study 2 was a phase II, open-label, single-arm, multicenter trial toevaluate the overall efficacy, safety, tolerability and the effect oftumor-based HER2 activation on the efficacy of pertuzumab in patientswith advanced ovarian cancer, in which their disease was refractory toor had recurred following prior chemotherapy. These women received IVinfusions of pertuzumab administered as a single agent over a 90-minuteperiod during the 1^(st) cycle of treatment at a fixed dose of 840 mg,followed by a 420 mg maintenance dose, delivered as a 30-minute infusionevery 3 weeks during subsequent treatment cycles. During the first andsecond treatment cycle, serum samples for determination of pertuzumabconcentrations were collected prior to the dose, 15 minutes followingthe end of the infusion, and at days 8 and 15. Additional serum samplesfor determination of pertuzumab concentrations were collected prior tothe dose and 15 minutes after the end of the IV infusion duringsubsequent treatment cycles.

Study 3 was a Phase II, open label, single-arm, multicenter randomizedstudy to evaluate the efficacy and safety of two different doses ofpertuzumab administered as a single agent in patients with metastaticbreast cancer with low expression of HER2. In first dose cohort,patients received pertuzumab as an IV infusion administered over a 90minute period as a 840 mg loading dose on the 1^(st) cycle, followed bya maintenance dose of 420 mg given every 3 weeks as a 30 minute IVinfusion during subsequent treatment cycles. In the second dose cohort,the patients received an IV infusion of pertuzumab as a 1050 mg doseover a 90-minute period on the 1^(st) cycle, and as a 1050 mg dose as a30-minute IV infusion every 3 weeks during subsequent treatment cycles.In study 3, serum samples for determination of pertuzumab concentrationswere collected prior to the dose, 15 minutes following the end ofinfusion, and on days 8 and 15 during the first two treatment cycles.

Additional serum samples for determination of pertuzumab concentrationswere collected prior to the dose and 15 minutes after the end ofinfusion during subsequent treatment cycles.

Drug Assay

Pertuzumab serum concentrations were determined by a validatedreceptor-binding, enzyme-linked, immunosorbent assay (ELISA). The assayused p185HER2 extracellular domain to capture pertuzumab from serumsamples. Bound pertuzumab was detected with mouse anti-humanFc-horseradish peroxidase (HRP) (Jackson ImmunoResearch Laboratories,Inc.), and tetramethyl benzidine (TMB) (KPL, Inc.) was used as thesubstrate for color development to quantify serum pertuzumab. The assayhas a minimum quantifiable concentration of 0.25 mcg/mL for pertuzumabin human serum.

Population Pharmacokinetic Analysis

Population non-linear mixed-effect modeling was performed using NONMEM™(Boeckmann and Beal NONMEM™ User Guide. San Francisco: NONMEM™ ProjectGroup, University of California, San Francisco (1994)) software (VersionV, Level 1.0) with NM-TRAN and PREDPP and the Compaq Visual Fortrancompiler (Version 6.5). Two different basic structural models, a one-and two-compartmental linear PK model with IV infusion, were fit toserum pertuzumab concentration-time data. The first-order conditionalestimation (FOCE) method with η-ε interaction was used throughout themodel-building procedure.

An exponential error model was used to describe the interindividualvariability for the PK parameters:

Pi={circumflex over (P)} _(i)exp(η_(iP))  (1)

A multiplicative covariate regression model was implemented as follows:

$\begin{matrix}{\hat{Pi} = {{\theta_{1}\left( \frac{X_{i}}{{med}(X)} \right)}^{\theta \; x}\left( {1 + {\theta_{D}D}} \right)}} & (2)\end{matrix}$

where η_(ip) denotes the proportional difference between the “true”parameters (Pi) of i^(th) individual patients and the typical value({circumflex over (P)}_(i)) in the population, adjusted for values ofcovariates equal to those of this individual patient. The η_(ip) is therandom effects with mean zero and variance ω². The θ_(X), and θ_(D) arethe regression coefficients to be estimated for continuous (e.g. WT) ordichotomous (e.g. SEX and RACE) covariates, respectively. Continuousvariables were centered on their median (med (X)) values, thus allowingθ₁ to represent the clearance estimate for the typical patient withmedian covariates. Dichotomous covariates were coded 0 or 1 (e.g., SEX=0for female, SEX=1 for male; RACE=O for Caucasian, and RACE=1 forothers). The residual variability was modeled as proportional-additiveerror model:C_(pij)=Ĉ_(pij) (1+ε_(ij,prop))+ε_(ij,add), where C_(pij) and Ĉ_(pij)are the j^(th) measured and model-predicted concentration, respectively,for i^(th) individual, and ε_(ij,prop) and ε_(ij,add) denote theproportional and additive residual intra-individual random errorsdistributed with zero means and variances σ_(prop) ² and σ_(add) ².

The relationships between structural model-based Bayesian estimates ofthe PK parameters and individual covariates were explored graphically.Based on preliminary exploratory analyses, the effect of each covariateon PK parameters was tested. Initially, the influence of covariates onindividual PK parameters (i.e., clearance and volume of the centralcompartment) was examined. Then, a full covariate model for theindividual PK parameters was constructed by incorporating thesignificant covariates into the model. A backward elimination processwas used to determine the final covariate model for each individual PKparameter by retaining only the significant covariates in the model.When highly correlated covariates had a similar pharmacological meaning(such as weight and BSA), only the most significant factor was retainedin the model. This final covariate model obtained for each PK parameterwas then combined to form a new full model and the final population PKmodel was elaborated using a backward elimination process.

Comparison of alternative structural models and construction of thecovariate model was based on the typical goodness-of-fit diagnosticplots and likelihood ratio test. When comparing alternative hierarchicalmodels, the differences in the value of the objective function isapproximately chi-square distributed with n degree of freedom (n is thedifference in the number or parameters between the full and the reducedmodel). This approximation has been shown to be reliable for theFOCE-INTERACTION estimation method (Wahlby et al. J PharmacokinetPharmacodyn 28:231-52 (2001)). To discriminate two hierarchical models,a difference in an objective function of greater than 7.9 (1 degree offreedom), which corresponds to a significance level of p<0.005, wasused.

The fraction of inter-individual variance (% variance) explained by thecovariates in the regression model for a given PK parameters (e.g., CL)was computed as follows:

$\begin{matrix}{{\% \mspace{14mu} {variance}} = {\left( \frac{\omega_{{CL},{BASED}}^{2} - \omega_{{CL},{FINAL}}^{2}}{\omega_{{CL},{BASE}}^{2}} \right) \times 100}} & (3)\end{matrix}$

Where ω² _(CL,BASED) and ω² _(CL,FINAL) represented inter-individualvariance of clearance in based and final PK model, respectively.

Population Pharmacokinetic Model Evaluation

The model evaluation in this study utilized a bootstrap resamplingtechnique to evaluate the stability of the final model and estimate theconfidence interval of parameters. This model evaluation techniqueconsists of first creating data sets using the bootstrap option in thesoftware package Wings for NONMEM™ (N Holford, Version 404, June 2003,Auckland, New Zealand) then obtaining parameter estimates for each ofthe replicate data sets. The results from 1000 successful runs wereobtained, and the mean and 2.5^(th) and 97.5^(th) percentiles (denotingthe 95% confidence interval) for the population parameters weredetermined and compared with the estimates of the original data.

In addition, a posterior predictive model checks were used to evaluatethe ability of the final model to describe the observed data (Yano etal. J Pharmacokinet Pharmacodyn 28:171-92 (2001); Gelman and Meng, Modelchecking and model improvement. In: Gilks W R, Richardson S,Spiegelhalter D J, eds. Markov Chain Monte Carlo in Practice. BocaRaton: Chapman & Hall/CRC, 189-202 (1996); Gelman et al. Bayesian DataAnalysis. Boca Raton: Chapman & Hall/CRC (2004). In these analyses, the2.5^(th), 5^(th), 10^(th), 25^(th), 50^(th) (median), 75^(th), 90^(th),and 95^(th) percentiles of the observed data were computed and selectedas the test statistics for the posterior predictive model check. Thefinal population PK model, including final fixed and random-effectparameters, was used to simulate 1000 replicates of the observed dataset and test statistics were computed from each of those simulateddataset. The posterior predictive distribution of test statistics fromthe simulated dataset was then compared with the observed teststatistics, and the p-value (p^(PPC)) can be estimated by calculatingthe proportion of cases in which test statistics from the simulated dataset exceed the realized value of observed test statistics according tothe following equation (Gelman and Meng, (1996) supra).

$\begin{matrix}{p^{PPC} = {\frac{1}{N}{\sum\limits_{i = 1}^{1000}\; {I\left( {{T\left( {y_{i}^{rep},\theta} \right)} \geq {T\left( {y,\theta_{i}} \right)}} \right)}}}} & (1)\end{matrix}$

Where I(.) is the indicator function which takes the value 1 when itsargument is true and 0 otherwise. T(y,θ) is a ‘realized value’ of theobserved test statistics because it is realized by the observed data y.T(y_(i) ^(rep), θ) is the test statistics from a simulated data set i(range from 1 to 1000) (Gelman and Meng, (1996) supra).

In addition, the 2.5^(th), 5^(th), 95^(th), and 97.5^(th) quantiles ofthe simulated data were calculated for each time points for individualpatients. The numbers of observed data that fell within the boundariesof the 2.5^(th) and 95.5^(th) quantiles (95% interval), 5^(th) and95^(th) quantiles (90% interval) of the pooled simulated data weredetermined.

Pertuzumab Exposures After Fixed, BSA-, and Weight-based Dosing

The final population PK model was used to determine the steady statetrough concentrations and exposure after fixed, BSA-, and weight-baseddosing. Serum concentration-time profiles and clearance of pertuzumabfor 1000 subjects were simulated for a fixed, BSA-based, or weight-baseddosing regimen using the final model with a dataset obtained bybootstrapping (with replacement) the original PK dataset. All simulatedsubjects received a 840 mg, 12.2 mg/kg, or 485 mg/m² iv infusion over 90min on Day 0, then a 420 mg, 6.1 mg/kg, or 242.5 mg/m′ infusion over 30min on Days 21, 42 and 63. Steady state trough concentrations obtainedon Day 84 (Css_(trough)) after different dosing regimens were thenassessed. In addition, the percent of subjects with steady state troughconcentrations below a target concentration (20 mcg/ml) after a fixed,BSA-based or weight-based dose were calculated. Simulated clearancevalues were used to determine the steady state average exposure(AUCss_(0-τ)) according to the following equation:

$\begin{matrix}{{AUCss}_{0 - \tau} = \frac{Dose}{CL}} & (4)\end{matrix}$

Results

Demographic Data.

The demographic characteristics of the patients included in this PKanalysis are listed in Table 2.

TABLE 2 Demographic characteristics of the patients included in theanalysis Median Range Age (years) 56.0 32.0-78.0 BSA* (m²) 1.731.40-2.53 Weight (kg) 69.0  45.0-150.6 Albumin (g/L) 39.2 21.0-52.0Alkaline phosphatase 107.0  39.0-367.0 (ALK) (IU/L) Number of Patients %Gender Male 8 5.2 Female 145 94.8 Race Caucasian 141 92.2 AfricanAmerican 3 2.0 Hispanic 4 2.6 Asian 3 2.0 Native Indian 0 0 Others 2 1.3*BSA, body surface area.

A total of 1458 pertuzumab serum concentration time points werecollected from 153 patients in the three studies. Of the total, 18patients were from the phase Ia trial, 60 from the phase II ovariancancer trial, and 75 from the phase II breast cancer trial. Thus, themajority (94.8%) of the patients in this analysis were female andaccounted for 1110 (76%) of the serum pertuzumab concentration data. Allsubjects had low HER2 expression tumor confirmed by FISH (fluorescencein situ hybridization) analysis and had good physical functional statusas indicated by an ECOG (Eastern Cooperative Oncology Group) performancestatus of either 0 or 1. The number of patients with missing covariateswas very low (4.6% for both height and BSA) and the missing covariateswere imputed with the median values. In 384 (20.8%) serum pertuzumabconcentration samples with only a documented sampling date, the samplingtime was imputed to occur at 12 noon. A sensitivity analysis conductedto assess the effects of these imputed times on the populationparameters estimates in the model revealed no significant influence.

Population PK Analysis.

A two-compartment model described the data better than one-compartmentmodel based on the change of objective function (δ=−736.2) anddiagnostic plots. A representative pertuzumab serum concentration-timeprofile fit to a one- and two-compartment model are illustrated in FIGS.9A-B. The inter-individual variability term (η) of K12 was removed fromtwo-compartmental models since the removal of this η term did not resultin a statistically significant increase (δ<7.88) in the objectivefunction. Therefore, only η_(CL), η_(Vc) and η_(k21) were retained inthe final base model.

The effect of the presence of a covariance term among η_(CL), η_(Vc) andη_(k21) was next assessed. Incorporation of a covariance terms amongη_(CL), η_(Vc) and η_(k21) improved the fit (δ=−23.2,df=3). However, thecovariance terms were found to be poorly estimated (% CV>100), have asmall estimated correlation (r_(CL-Vc)=0.37; r_(CL-K21)=0.27;r_(Vc-K21)=0.42), and have little influence on parameter estimations(data not shown). Therefore, the covariance terms were not retained forcovariate effect model building. In an exploratory analysis using thefinal base model, no apparent relationships between potential covariatesand η_(k21) were identified. Therefore, covariate effect on η_(k21) wasnot examined during the development of the final model with covariates.

For the final model with covariates, predicted versus observedpertuzumab serum concentrations and weighted residuals versus predictedserum concentration plots are shown in FIGS. 10A-B. In the final model,serum albumin (ALB), body weight (BW) and serum alkaline phosphatase(ALKP) were the most significant covariates explaining interindividualvariability for pertuzumab clearance (CL). BSA was the most significantcovariate explaining interindividual variability of pertuzumab centralcompartment volume of distribution (Vc). Incorporation of covarianceterms among η_(CL), η_(Vc) and η_(k21) improved the fit (δ=−14.0,df=3).However, the estimated correlation was not large (r_(CL-Vc)=0.45;r_(CL-K21)=0.28; r_(Vc-K21)=0.39) and the parameter estimates were notinfluenced (data not shown). Hence, the covariance terms were notincluded in the final model. The final model was illustrated as follows:

$\begin{matrix}{{CL} = {\theta_{CL} \times \left( \frac{WT}{69} \right)^{\theta_{WT\_ CL}} \times \left( \frac{ALB}{39.2} \right)^{\theta_{ALB\_ CL}} \times \left( \frac{ALKP}{107} \right)^{\theta_{ALKP\_ CL}}}} & (4) \\{{Vc} = {\theta_{Vc} \times \left( \frac{BSA}{1.72} \right)^{\theta_{BSA\_ Vc}}}} & (5)\end{matrix}$

The parameter estimates of the final model are summarized in Table 3.

TABLE 3 Parameter Estimates of the Final Population PharmacokineticModel and the Stability of the Parameters Using a Bootstrap ValidationProcedure 1000 bootstrap Original Data Set Replicates Estimate (%RSE)^(a) Mean (95% CI) Structural Model CL (L/day) 0.214 (3.1)  0.214(0.201, 0.228) Vc (L) 2.740 (1.9)  2.739 (2.640, 2.840) K12 (day⁻¹)0.203 (16.6) 0.220 (0.159, 0.416) K21 (day⁻¹) 0.258 (15.6) 0.275 (0.203,0.480) Inter-individual Variability CL % CV  31.1 (11.0) 30.6 (27.0,34.1)  Vc % CV  16.2 (20.3) 16.0 (12.7, 19.2)  K₂₁% CV  25.2 (37.6) 24.1(11.4, 33.6)  Covariate Model ALB on CL −1.010 (18.4)   −1.019 (−1.420,−0.632) (θ_(ALB) _(—) _(CL)) WT on CL (θ_(WT) _(—) _(CL)) 0.587 (19.3)0.589 (0.372, 0.826) ALKP on CL 0.169 (29.5) 0.170 (0.067, 0.258)(θ_(ALKP) _(—) _(Vc)) BSA on Vc (θ_(BSA Vc)) 1.160 (12.2) 1.151 (0.890,1.451) Residual Variability Proportional error 0.037 (19.4) 0.037(0.030, 0.045) σ² _(prop) Additive error, σ_(prop), 2.265 (77.8)  2.24(0.002, 4.160) mcg/mL ^(a)% RSE: percent relative standard error of theestimate = SE/parameter estimate × 100

The CL of serum pertuzumab in the analysis population was estimated tobe 0.214 L/day and the Vc was 2.74 L. The K₁₂ and K₂₁ were 0.203 and0.258 days⁻¹, respectively. Interindividual variability for CL and Vc inthe final model, calculated as the square root of interindividualvariance (ω²) and expressed as % CV, are 31.1% and 16.2%, respectively,compared to 38.0% and 20.8% for the base model without covariates. Thecovariate effect of ALB, WT, and ALKP in the final model thereforeexplained about 33% of the interindividual variance for CL. However,weight alone explained only 8.3% of inter-patient variability for CL.The covariate effect of BSA explained about 39% of interindividualvariance for Vc in the final model. The dependency of CL on WT and Vc onBSA with the base model is accounted for in the final model as shown inFIGS. 11A-B. The estimated t_(1/2), and t_(1/2β) were 1.4 and 17.2 days,respectively.

Model Evaluation.

From the original dataset, 1000 successful bootstrap runs were obtainedand compared to the original observed data. Mean population PK estimatesobtained from the bootstrap procedure were similar to the parameterestimates of the original dataset (Table 3), indicating that thedeveloped model was stable. The 95% confidence intervals for thefixed-effect parameters were narrow, which indicated good precision.

A posterior predictive model check was used to evaluate the ability ofthe final model to describe the observed data. The final populationpharmacokinetic model, including final fixed and random-effectparameters, was used to simulate 1000 replicates. The test statisticswere then computed for each of those 1000 simulated dataset. FIGS. 12A-Fdisplay histograms of the 1000 simulated values of selected teststatistics, with the “realized value” of the observed test statisticsindicated by vertical line. The posterior predictive distributions wereclose to the observed values with the estimated p-values greater than0.05 for each test statistics. In addition, the percents of observedpertuzumab concentrations within 90% and 95% quantile range of thepooled simulated data were 89.3 and 94.7%, respectively. These resultssuggested that the model was able to describe and predict the datareasonably well.

Pertuzumab Exposures After Fixed, BSA-, and Weight-based Dosing.

Predicted pertuzumab steady-state trough serum concentrations on day 84(C_(ss,trough)) were estimated for 1000 simulated subjects bootstrappedfrom the original PK dataset and the final model using a fixed,weight-based, or BSA-based dose according to the dose schedules outlinedin the methods section. These data showed that with weight-based andBSA-based dosing, population variability of C_(ss,trough) decreased by6.17 and 5.76%, respectively, when compared to fixed dosing (FIG. 13 andTable 4).

TABLE 4 Predicted Pertuzumab Steady State Trough Concentration (Day 84)After a Fixed, Weight or BSA-based Dose for 1000 Simulated SubjectsBootstrapped from Original PK Dataset According to the Final Model Csstrough Css trough Css trough (mcg/ml) (mcg/ml) Weight- (mcg/ml) BSA-Fixed Dose based Dose based Dose Minimum 2.68 2.39 2.54 5^(th)Percentile 16.56 16.32 16.86 Median 51.87 51.81 52.48 Mean 56.37 56.0856.44 95^(th) Percentile 115.38 110.46 112.14 Maximum 209.67 179.06192.00 % CV 54.05 52.62 52.40 Variance 928.21 870.93 874.71 % VarianceChanged — −6.17 −5.76 from Fixed Dose ^(a) Percent of Subjects 8.3 8.78.3 with C_(sstrough) ≦ 20 mcg/ml ^(a) Percent variance changed fromfixed dose was calculated using the following equation:

${{Percent}\mspace{14mu} {variance}\mspace{14mu} {change}} = {\frac{{Variance}_{{{WT}\mspace{14mu} {or}\mspace{14mu} {BSA}} - {{based}\mspace{14mu} {dose}}} - {Variance}_{{fixed}\mspace{14mu} {dose}}}{{Variance}_{{fixed}\mspace{14mu} {dose}}} \times 100}$

The percentage of subjects with C_(sstrough) below a target serumconcentration of 20 mcg/ml were similar, with values of 8.3%, 8.7%, and8.3% for fixed, weight-based, or BSA-based dosing, respectively (Table4). Similar results were obtained from the analysis of pertuzumab serumsteady state AUC_(ss) _(0-τ) for 1000 simulated subjects, and weight-and BSA-based dosing only reduced the population variability by 2.2 and4.2%, respectively, when compared to fixed dosing. The same simulateddataset was used to determine C_(ss,trough) after a fixed dose, weight-,and BSA-based dose for populations with extreme weight (i.e., WT≦10^(th)and ≧90^(th) percentile) (FIGS. 14A-B). Median pertuzumab C_(ss,trough)for population with WT less than or equal to 10^(th) percentile were72.3 (range: 8.7 to 166.5), 52.8 (range: 6.8 to 125.7) and 63.2 (range:7.8 to 150.1) mcg/ml for a fixed dose, weight-, and BSA-based dose,respectively. The percentage of subjects in population withC_(ss,trough) below a target serum concentration of 20 mcg/ml were 5.4%,12.6%, and 9.0% for fixed, weight-based, and BSA-based dosing,respectively. Median pertuzumab C_(ss,trough) for population with WTgreater than or equal to 90^(th) percentile were 42.1 (range: 7.0 to119.8), 62.8 (range: 14.4 to 167.3) and 52.9 (range: 10.2 to 133.3)mcg/ml for a fixed dose, weight-, and BSA-based dose, respectively. Thepercentages of subjects in this population with C_(ss,trough) below atarget serum concentration of 20 mcg/ml were similar, with values of7.4%, 2.8%, and 5.6% for fixed, weight-based, or BSA-based dosing,respectively. Similar results were obtained for the analysis ofpertuzumab serum steady state AUC_(ss) _(0-τ) of these subgroups from1000 simulated subjects.

Discussion

Typically, humanized IgG monoclonal antibodies and cytotoxic smallmolecule drugs in oncology have been administered on a weight-based(mg/kg) or BSA-based dose basis. Pertuzumab has undergone testing in theclinic with a phase Ia trial in patients with advanced cancers and inphase II trials in patients with ovarian, breast, lung, and prostatecancer. Pertuzumab was dosed on a weight-basis (mg/kg) in a Phase Itrial, and then initiated using a fixed dose in a phase II trials. Usingdemographic and serum pertuzumab concentration-time data collected inthese three trials, a population PK model with predictive covariates forpertuzumab PK was built herein. This model was then used to examine thesteady-state concentrations after fixed dosing, weight- and BSA-baseddosing methods.

Pertuzumab PK obtained from this analysis was very similar to thosereported for other humanized monoclonal IgG1 drugs used in oncology(Harris et al. Proc Am Soc Clin Oncol (Abstract #488) 21:123a (2002);Leyland-Jones et al. J Clin Oncol 21:3965-71 (2003); and Lu et al. ClinPharmacol Ther 75:91 (2004)).

A linear 2-compartment linear PK model best describe the data and in thefinal model pertuzumab CL was 0.214 L/day. Typical Vc of pertuzumab was2.74 L or approximately 40 ml/kg, which is equal to human plasma volumeand was consistent with values reported for other monoclonal IgG1 drugs(Harris et al. (2002), supra; and Lu et al. (2004), supra). PertuzumabCL was significantly affected by body weight and serum concentrations ofalbumin and alkaline phosphatase, while Vc was significantly influencedby BSA. The effect of gender on the pertuzumab PK cannot be assessedbecause of the small number of male subjects (5.2%) included in theanalysis. The results from a bootstrap procedure and posterior modelchecking suggested that the final model was stable and able to describeand predict the data reasonably well.

The effect of weight on CL and BSA on Vc suggested that pertuzumab mightbe dosed based on either body weight or BSA. However, the covariateeffect of weight alone and BSA alone in the model only explained about8.3% and 40% of the inter-individual effect of CL and Vc, respectively.This suggested that while weight is a predictor of CL and BSA is apredictor for Vc, the effect of weight and BSA on pertuzumab exposuresafter dosing might be measurable but not highly contributory.

Therefore, the next step assessed the impact of the various dosingmethods on the pertuzumab exposures using simulations. In 1000 subjectsbootstrapped from the original data set, weight-based or BSA-baseddosing were found to decrease population variability of simulated steadystate trough serum concentrations on day 84 by only 6.2 and 5.8%,respectively, when compared to fixed dosing. In addition, thepercentages of subjects with predicted steady-state trough serumconcentrations below a selected target of 20 mcg/mL were similar withall three dosing methods. Similar results were obtained from thesubgroup analysis in population with extreme body weight (i.e.,WT≦10^(th) and ≧90^(th) percentile).

Hence, it is concluded that pertuzumab PK is related to WT and BSA.However, the WT and BSA explained only a small percent of theinter-individual variability of CL and Vc, and WT- and BSA-based dosingdo not seem to improve the predictability of pertuzumab steady stateexposures. It is recommended to apply fixed-dosing regimens forpertuzumab in cancer patients.

The present invention is believed to represent the first disclosure of acritical assessment of the impact of weight- or BSA-based dosing of ahumanized IgG1 monoclonal antibody on steady state drug concentrationsin cancer patients. Implementation of flat-fixed dosing has severalsignificant patient care and economic implications: i) lower costs dueto greater efficiency in manufacturing, storing and shipping of singleunit dose, ii) efficient preparation of a single dose in pharmacies andhospitals without the need for patient individualization, iii) greaterefficiency in physician prescribing of single unit dose, and iv) lowerlikelihood of patient receiving wrong dose due to dose calculationerrors. Although humanized antibodies are typically dosed by weight orBSA, the analyses herein demonstrate the feasibility of administratingthe HER antibody pertuzumab using a fixed dose in cancer patients.

What is claimed is:
 1. A method for treating cancer comprisingadministering one or more fixed dose(s) of a HER antibody to a humanpatient in an amount effective to treat the cancer.
 2. The method ofclaim 1 wherein the antibody binds to a HER receptor selected from thegroup consisting of EGFR, HER2, and HER3.
 3. The method of claim 2wherein the antibody binds to HER2.
 4. The method of claim 3 wherein theHER2 antibody binds to Domain II of HER2 extracellular domain.
 5. Themethod of claim 1 wherein the antibody binds to a junction betweendomains I, II and III of HER2 extracellular domain.
 6. The method ofclaim 1 wherein the HER antibody inhibits heterodimerization of HER2with EGFR or HER3.
 7. The method of claim 1 wherein the HER antibodycomprises the variable light and variable heavy amino acid sequences inSEQ ID Nos. 3 and 4, respectively.
 8. The method of claim 1 wherein theHER antibody is pertuzumab.
 9. The method of claim 1 wherein the fixeddose is in the range from about 20 mg to about 2000 mg of the HERantibody.
 10. The method of claim 9 wherein the fixed dose is selectedfrom the group consisting of approximately 420 mg, approximately 525 mg,approximately 840 mg, and approximately 1050 mg of the HER antibody. 11.The method of claim 10 wherein the fixed dose is 420 mg of the HERantibody.
 12. The method of claim 10 wherein the fixed dose is 840 mg ofthe HER antibody.
 13. The method of claim 10 wherein the fixed dose is1050 mg of the HER antibody.
 14. The method of claim 10 wherein thefixed dose is 525 mg of the HER antibody.
 15. The method of claim 1wherein a fixed dose of the HER antibody is administered to the patientapproximately every week, approximately every 2 weeks, approximatelyevery 3 weeks, or approximately every 4 weeks.
 16. The method of claim15 wherein a fixed dose of the HER antibody is administered to thepatient approximately every 3 weeks.
 17. The method of claim 10comprising administering a loading dose of approximately 840 mg of theHER antibody followed by one or more maintenance doses of approximately420 mg of the HER antibody.
 18. The method of claim 17 wherein themaintenance doses are administered approximately every 3 weeks.
 19. Themethod of claim 10 comprising administering a loading dose ofapproximately 1050 mg of the HER antibody followed by one or moremaintenance doses of approximately 525 mg of the HER antibody.
 20. Themethod of claim 19 wherein the maintenance doses are administeredapproximately every 3 weeks.
 21. The method of claim 1 wherein the HERantibody is a naked antibody.
 22. The method of claim 1 wherein the HERantibody is an intact antibody.
 23. The method of claim 1 wherein theHER antibody is an antibody fragment comprising an antigen bindingregion.
 24. The method of claim 1 wherein the HER antibody is ahumanized or human IgG1 antibody.
 25. The method of claim 1 wherein thecancer displays HER expression, amplification, or activation.
 26. Themethod of claim 1 wherein the cancer is ovarian, peritoneal, orfallopian tube cancer.
 27. The method of claim 1 wherein the cancer ismetastatic breast cancer (MBC).
 28. The method of claim 1 wherein thecancer is non-small cell lung cancer (NSCLC).
 29. The method of claim 1wherein the cancer is prostate cancer.
 30. The method of claim 1 whereinthe cancer is colorectal cancer.
 31. The method of claim 1 comprisingadministering a second therapeutic agent to the patient.
 32. The methodof claim 31 wherein the second therapeutic agent is selected from thegroup consisting of chemotherapeutic agent, different HER antibody,antibody directed against a different tumor associated antigen,anti-hormonal compound, cardioprotectant, cytokine, EGFR-targeted drug,anti-angiogenic agent, tyrosine kinase inhibitor, COX inhibitor,non-steroidal anti-inflammatory drug, farnesyl transferase inhibitor,antibody that binds oncofetal protein CA 125, HER2 vaccine, another HERtargeting therapy, Raf or ras inhibitor, doxorubicin HCL liposomeinjection, topotecan, taxane, dual tyrosine kinase inhibitor, TLK286,EMD-7200, a medicament that treats nausea, a medicament that prevents ortreats skin rash or standard acne therapy, a body temperature-reducingmedicament, and a hematopoietic growth factor.
 33. The method of claim32 wherein the second therapeutic agent is a chemotherapeutic agent. 34.The method of claim 33 wherein the chemotherapeutic agent is anantimetabolite chemotherapeutic agent.
 35. The method of claim 34wherein the antimetabolite chemotherapeutic agent is gemcitabine. 36.The method of claim 31 wherein the second therapeutic agent istrastuzumab, erlotinib HCL, or bevacizumab.
 37. A method of treatingcancer in a human patient comprising administering at least one fixeddose of pertuzumab to the patient, wherein the fixed dose is selectedfrom the group consisting of approximately 420 mg, approximately 525 mg,approximately 840 mg, and approximately 1050 mg of pertuzumab.
 38. Themethod of claim 37 wherein a fixed dose of pertuzumab is administered tothe patient approximately every 3 weeks.
 39. The method of claim 37wherein the cancer is selected from the group consisting of ovariancancer, peritoneal cancer, fallopian tube cancer, metastatic breastcancer (MBC), non-small cell lung cancer (NSCLC), prostate cancer, andcolorectal cancer.
 40. The method of claim 37 wherein the fixed dose isselected from the group consisting of 420 mg, 525 mg, 840 mg, and 1050mg of pertuzumab.
 41. An article of manufacture comprising a vialcontaining a fixed dose of a HER antibody, wherein the fixed dose isselected from the group consisting of approximately 420 mg,approximately 525 mg, approximately 840 mg, and approximately 1050 mg ofthe HER antibody.
 42. The article of manufacture of claim 41 wherein theHER antibody is pertuzumab.
 43. The article of manufacture of claim 41wherein the fixed dose is selected from the group consisting of 420 mg,525 mg, 840 mg, and 1050 mg of the HER antibody.
 44. The article ofmanufacture of claim 41 further comprising a package insert instructingthe user to administer the fixed dose to a cancer patient.
 45. Thearticle of manufacture of claim 44 wherein the cancer is selected fromthe group consisting of ovarian cancer, peritoneal cancer, fallopiantube cancer, metastatic breast cancer (MBC), non-small cell lung cancer(NSCLC), prostate cancer, and colorectal cancer.
 46. The article ofmanufacture of claim 44 wherein the package insert further instructs theuser to administer the fixed dose to a cancer patient whose cancerdisplays HER expression, amplification, or activation.
 47. The articleof manufacture of claim 41 comprising two vials, wherein a first vialcontains a fixed dose of approximately 840 mg of pertuzumab, and asecond vial contains a fixed dose of approximately 420 mg of pertuzumab.48. The article of manufacture of claim 41 comprising two vials, whereina first vial contains a fixed dose of approximately 1050 mg ofpertuzumab, and a second vial contains a fixed dose of approximately 525mg of pertuzumab.